WO2005039588A2

WO2005039588A2 - Methods for determining the risk of developing liver and lung toxicity

Info

Publication number: WO2005039588A2
Application number: PCT/EP2004/011921
Authority: WO
Inventors: Kenneth Wayne Culver; David Parkinson; Diane Mccarthy
Original assignee: Novartis Ag; Novartis Pharma Gmbh
Priority date: 2003-10-22
Filing date: 2004-10-21
Publication date: 2005-05-06
Also published as: WO2005039588A3

Abstract

Methods are disclosed for, predicting the probability or likelihood that an individual will develop hepatotoxicity or lung toxicity when treated with a therapeutic or study agent that has the potential to cause liver or lung toxicity and methods for detecting at an early stage the development of hepatoxicity during treatment with a potentially hepatotoxic therapeutic or study agent. These methods involve determining the level of serum amyloid A or the gene expression products of the SERPINA3 gene, either mRNA or protein, in the body fluids or tissues of the individual. Kits for the performance of these assays are also provided. A method is disclosed using [11(R)-4-[(1-phenylethyl)amino]-7H-pyrrolo [2,3-d] pyrimidin -6-yl] -phenol in the manufacture of a medicament for the treatment of proliferative disease.

Description

METHODS FOR DETERMINING THE RISK OF DEVELOPING LIVER AND LUNG TOXICITY

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[01] This invention relates generally to the analytical testing of tissue samples in vitro, and more particularly to methods for distinguishing patients who will develop or are developing liver and/or lung toxicity from those who will not.

[02] These methods involve the use of genomic, proteomic or metabolomic techniques to measure or determine the presence and level, in body fluids or tissues, of certain classes of biochemicals and gene expression products, including proteins. The level of these gene expression products can be correlated with the likelihood of the development of liver or lung toxicity or the presence of such toxicity. These methods have utility as diagnostic biomarkers or theranostics to monitor patients during treatment or drug trials. Kits for performing these assays are also provided.

DESCRIPTION OF THE RELATED ART

[03] All drugs have undesirable effects as well as the desired therapeutic ones and the spectrum of such effects may be broad and ill-defined. In therapeutics usually only one of the numerous effects produced by a drug is sought as the primary goal of treatment. Most other effects are referred to as undesirable effects and are either side effects or toxic effects. Toxic effects may be produced by the direct action of the drug or may be due to metabolites of the drug produced by enzymes, light or by reactive oxygen species. [04] Toxic effects may be classified as pharmacological, pathological, genotoxic (alterations in DNA) and as dose dependent or dose independent. An example of a pharmacologic toxicity is the excessive depression of the central nervous system (CNS) by barbiturates; an example of a pathological toxic effect is the hepatic injury caused by acetaminophen and an example of a genotoxic effect is a neoplasm produced by a nitrogen mustard. Dose dependent toxic effects become more pronounced as the administered dose of the drug increases and often resolve when the drug is withdrawn while dose independent toxic effects may be provoked by even a small dose and low levels of the drug or toxin and may continue or worsen even if the toxic agent is then withdrawn. [05] Local toxicity is the effect that occurs at the site of first contact between the biological system and the toxicant. Local effects can be caused by ingestion of caustic substances or inhalation of irritant materials. Systemic toxicity requires absorption and distribution of the toxicant, most drug substances produce systemic toxic effects. [06] Most systemic toxicants affect one or a few organs predominantly. The target organ of toxicity is not necessarily the site of accumulation of the chemical. The CNS is involved in systemic toxicity most frequently as many compounds with prominent effects elsewhere also affect the brain. Next in order of frequency in systemic toxicity are the circulatory system: the blood and haematopoietic system; visceral organs such as the liver, kidney, and lung and the skin. The liver and lung are of particular concern since the liver is the site of much of the detoxification reactions that serve to rid the body of chemicals and drugs by means of a variety of metabolic enzyme systems, such as the P450 system. [07] In addition, damage to the liver or the lung can cause rapidly fatal organ dysfunction and may occur in the context of the therapeutic administration of a drug to treat a specific disease or may occur in the context of the administration of an experimental drug or unapproved drug for the purpose of determining the safety of efficacy of the drug (a drug study).

[08] The central role of the liver in drug metabolism results in this organ being exposed to a large variety of potentially toxic chemical agents and metabolites. The manifestations of toxic and drug-induced liver disease constitute a broad spectrum of clinical, laboratory and histopathological changes.

[09] Some of the early and fairly sensitive indicators of toxic effects on the liver are elevations in the blood levels of enzymes normally contained in liver cells. These enzymes are released into the blood when the liver cells are damaged or destroyed by a toxic agent. Thus, routine surveillance for impending liver damage often involves determining the blood levels of enzymes such as aspartate aminotransferase (AST) or alanine aminotransferase (ALT). However, the elevation of the levels of these enzymes may not occur rapidly with the development of toxic effects to the liver and once the damage is measurable by these means it may be too late to prevent the development of severe and even life threatening liver damage. (See, e.g., Goodman & Gilman's The Pharmacologic Basis of Therapeutics, Ninth Edition, Molinoff P.B. and Ruddon R.W., Editors, McGraw-Hill New York 1996 and Cecil Text book of Medicine, 21^st Edition, Goldman L. and Claude Bennett J. Editors, W.B. Saunders Company, Philadelphia 2000).

[10] Thus there is a need for more sensitive and rapidly responsive tests for the development of toxic effects on the liver or lungs and other organs of drugs being tested in humans or for drugs being administered to a patient to achieve a therapeutic effect and for tests to determine, prior to administration of a drug, the likelihood that a specific individual or patient will develop potentially serious toxic effects if the drug is given.

SUMMARY OF THE INVENTION

[11] This invention provides a method for determining, prior to treatment, which individuals will develop dose-independent hepatotoxicity or lung toxicity when treated with a therapeutic or study agent; comprising: a) obtaining a sample of tissue or body fluid from the said individual prior to treatment; b) determining the level of production of the gene expression product (or fragment of the gene expression product) of the SERPINA3 gene (see SEQ ID NO:1, for nucleotide and SEQ ID NO:2 for polypeptide) in the said sample of tissue or body fluid to obtain a first value; c) determining the average value and the standard deviation in the values of the production of the said gene expression product of the SERPINA3 gene in at least 10 similar samples of tissue or body fluid from at least ten similar individuals to obtain a second value and standard deviation; d) comparing the first value with the second value; e) determining that the said individual is in a high risk group for developing dose-independent hepatotoxicity if the first value, determined in (a) is more than two standard deviations below the second value determined in (b); and f) determining that the said individual is in a low risk group if the first value determined in (a) is less than two standard deviations below the second value determined in (b) or is equal to or greater than the second value determined in (b). wherein the said sample of tissue or body fluid is selected from the group consisting of a tissue biopsy, blood, serum, plasma, lymph, ascitic fluid, cystic fluid, urine, cerebro-spinal fluid (CSF), salvia or sweat. Sequence variants of SERPIN A3 are known to those of skill in the art; these variants are included in the scope of the invention.

[12] In some embodiments this invention provides a method as above, wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the polypeptide expression product of the SERPINA3 gene in the said sample of tissue or body fluid, wherein the said polypeptide expression product of the SERPINA3 gene is the protein alpha-1 anti-chymotrypsin (SEQ ID NO:2).

[13] In addition this invention provides a method wherein the step of determining the level of production of the gene expression product of the SERP1NA3 gene in the said sample of tissue or body fluid is preformed by measuring the level of the polypeptide gene expression product by means of mass spectrometry. In some preferred embodiments the mass spectrometry technique used is Surface Enhanced Laser Desorbtion/lonization Time Of Flight Mass Spectrometry (SELDI-TOF-MS) or is Matrix-Assisted Laser Desorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLDI-TOF-MS) [14] In addition, this invention provides a method wherein the presence of the polypeptide expression product of the SERPINA3 gene protein (SEQ ID NO:2) is detected using a reagent which specifically binds with the said polypeptide and may be a labelled probe specific for the protein. Or may be selected from the group consisting of an antibody, such as a monoclonal antibody, an antibody derivative and an antibody fragment. [15] In other embodiments this invention provides a test kit for use in determining which individuals will develop dose-independent hepatotoxicity when treated with a therapeutic or study agent; comprising the reagent described above in a container suitable for contacting the said body fluid, with instructions for interpreting the results. The reagent may comprise an antibody including a monoclonal antibody, wherein said antibody specifically binds with the polypeptide expression product of the SERPINA3 gene.

[16] In addition, this invention provides a method for determining, prior to treatment, which individuals will develop dose-independent hepatotoxicity when treated with a therapeutic or study agent; wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the mRNA expression product of the SERPINA3 gene in the said sample of tissue or body fluid and wherein the level of expression of the mRNA expression product of the SERPINA3 gene is determined by techniques selected from the group consisting of: hybridization to a nucleotide array, Northern blot analysis, RT-PCR and real time quantitative PCR. In using these methods the therapeutic or study agent may be an epidermal growth factor receptor inhibitor (EGFRI) including but not limited to PKI166 or may be an oxidizing drug. [17] In alternate embodiments this invention provides a method for monitoring the progression or development of hepatotoxicity in an individual being treated with a therapeutic or study agent, the method comprising: a) obtaining a pre-treatment sample of body fluid or tissue from the individual prior to administration of the agent, b) detecting a level of expression of the protein SAA (se SEQ ID NO: 3 for nucleotide and SEQ ID NO:4 for polypeptide) in the said body fluid or tissue sample; c) obtaining one or more post- administration samples of body fluid or tissue from the subject during or following treatment with the said therapeutic agent; d) detecting a level of expression of the protein SAA (SEQ ID NO:4) in one or more post-administration sample or samples; e) comparing the level of expression of protein SAA detected in (b) to the level detected in (d); and f) determining from the comparison of the two or more levels of SAA protein the likelihood that the individual is developing hepatotoxicity and adjusting the administration of the agent accordingly. Sequence variants of SAA are known to those of skill in the art; these variants are included in the scope of the invention. The sample may be selected from the group consisting of; a tissue biopsy, blood, serum, plasma, lymph, ascitic fluid, cystic fluid, urine, cerebro-spinal fluid (CSF), salvia or sweat.

[18] In addition, the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment may be performed by determining the level of the mRNA expression product of the SERPINA3 gene (SEQ ID NO:1) in the said sample of tissue or body fluid by techniques selected from the group consisting of Northern blot analysis, hybridization to a nucleotide array, RT-PCR and real time quantitative PCR.

[19] In addition, this invention includes methods for monitoring the progression or development of hepatotoxicity in a subject having, or at risk of having, hepatotoxicity during or after treatment with a therapeutic or study agent, comprising measuring a level of expression of the gene expression product of the SAA gene over time in a sample of bodily fluid or tissue obtained from the subject during treatment, wherein an increase in the level of expression of the said protein over time is indicative of the development of hepatotoxicity in the subject. This measurement may be performed by measuring the level of production of the gene expression product of the SAA gene protein SAA over time such as by measuring the level of SAA protein in a sample of bodily fluid or tissue obtained from the subject by means of mass spectrometry methods. In preferred embodiments these measurements would be performed by means of mass spectrometry methods such as Surface Enhanced for Laser Desorbtion/lonization Time Of Flight Mass Spectrometry (SELDI-TOF-MS) or Matrix-Assisted Laser Desorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLDI- TOF-MS).

[20] In alternate embodiments, this measurement may be performed by using a reagent which specifically binds with the said polypeptide. This reagent may be selected from the group consisting of an antibody, a monoclonal antibody, an antibody derivative and an antibody fragment.

[21] In addition this invention includes test kits for use in determining which individuals are developing dose-independent hepatotoxicity when treated with a therapeutic or study agent; comprising the reagent above in a container suitable for contacting the said body fluid with instructions for interpreting the results. Such reagent may comprise an antibody, wherein said antibody specifically binds with the protein SAA (SEQ ID NO:4). [22] In addition, this invention includes methods wherein the step of determining the level of production of the gene expression product of the SAA gene in the said sample of tissue or body fluid is performed by determining the level of the mRNA expression product of the SAA gene in the said sample of tissue or body fluid. This may be determined by techniques selected from the group consisting of: hybridization to a nucleotide array, Northern blot analysis, RT-PCR and real time quantitative PCR.

[23] In various embodiments this invention includes methods wherein the therapeutic or study agent is an epidermal growth factor receptor inhibitor (EGFRI), including but not limited to PKI166 or the agent may be any oxidizing drug.

[24] In alternate embodiments, this invention includes methods for determining, prior to initiation of treatment, which individual(s) should be included in a study of a therapeutic or study agent; comprising: a) obtaining a sample of tissue or body fluid from the said individual(s) prior to treatment; b) determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid to obtain a first value; c) determining the average value and the standard deviation in the values of the production of the said gene expression product of the SERPINA3 gene in at least 10 similar samples of tissue or body fluid from at least ten similar individuals to obtain a second value and standard deviation; d) comparing the first value with the second value; e) determining that the said individual is in a high risk group for developing dose-independent hepatotoxicity, and should not be included in the treatment or study, if the first value, determined in (a) is more than two standard deviations below the second value determined in (b); and f) determining that the said individual is in a low risk group and may be included in the treatment or study, if the first value determined in (a) is less than two standard deviations below the second value determined in (b) or is equal to or greater than the second value determined in (b).

[25] The above method may involve determining the level of production of the gene expression product of the SERPINA3 gene (SEQ ID NO:1) in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the polypeptide expression product of the SERP1NA3 gene (SEQ ID NO:2) in the said sample of tissue or body fluid, such as a tissue biopsy, blood, serum, plasma, lymph, ascitic fluid, cystic fluid, urine, cerebro-spinal fluid (CSF), salvia or sweat. The said polypeptide expression product of the SERPINA3 gene is the protein alpha-1 anti-chymotrypsin. [26] The above method may involve the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid by measuring the level of the polypeptide gene expression product by means of mass spectrometry, including Surface Enhanced for Laser Desorbtion/lonization Time Of Flight Mass Spectrometry (SELDI-TOF-MS) and Matrix-Assisted Laser Desorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLDI-TOF-MS)

[27] In addition, in other embodiments of this invention the presence of the polypeptide expression product of the SERPINA3 gene protein is detected using a reagent which specifically binds with the said polypeptide. This reagent may be a labelled probe specific for the protein, including but not limited to; an antibody, an aptamer, an antibody derivative and an antibody fragment and including monoclonal antibodies.

[28] In other embodiments this invention includes a test kit for use in determining which individuals will develop dose-independent hepatotoxicity when treated with a therapeutic or study agent and should not be included in a study or use of that therapeutic agent; comprising the reagent above in a container suitable for contacting the said body fluid with instructions for interpreting the results. This reagent may be an antibody wherein said antibody specifically binds with the polypeptide expression product of the SERPINA3 gene. [29] In separate embodiments, this invention includes a method for monitoring the progression or development of hepatotoxicity in an individual being treated with a therapeutic or study agent, the method comprising: a) obtaining a pre-treatment sample of body fluid or tissue from the individual prior to administration of the agent; b) detecting a level of expression of the protein SAA in the said body fluid or tissue sample; c) obtaining one or more post-administration samples of body fluid or tissue from the subject during or following treatment with the said therapeutic agent; d) detecting a level of expression of the protein SAA in one or more post-administration sample or samples; e) comparing the level of expression of protein SAA detected in (b) to the level detected in (d); and f) determining from the comparison of the two or more levels of SAA protein the likelihood that the individual is developing hepatotoxicity and adjusting the administration of the agent accordingly.

[30] In the above method the said sample of tissue or body fluid may be selected from the group consisting of; a tissue biopsy, blood, serum, plasma, lymph, ascitic fluid, cystic fluid, urine, cerebro-spinal fluid (CSF), salvia or sweat.

[31] In some embodiments, the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the mRNA expression product of the SERPINA3 gene in the said sample of tissue or body fluid. This may be performed by use of Northern blot analysis, RT-PCR, hybridization to a nucleotide array and real time quantitative PCR.

[32] In other embodiments, this invention provides methods for determination of when treatment with a therapeutic or study agent should be discontinued in a subject at risk of having, hepatotoxicity during or after treatment with a therapeutic or study agent, comprising measuring a level of expression of the protein SAA over time in a sample of bodily fluid or tissue obtained from the subject during treatment, wherein an increase in the level of expression of the said protein over time is indicative of the development of hepatotoxicity in the subject and determines that the agent should be discontinued. The step of determining the level of expression of the protein SAA in the said sample of tissue or body fluid may, in preferred embodiments, be performed by measuring the level of the protein SAA by means of mass spectrometry, including, but not limited to Surfaces Enhanced for Laser Desorbtion/lonization Time Of Flight Mass Spectrometry (SELDI-TOF-MS) and Matrix- Assisted Laser Desorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLDI-TOF-MS) [33] In other embodiments the presence of the protein SAA is detected using a reagent which specifically binds with the said polypeptide. This reagent may be an antibody, an aptamer, an antibody derivative and an antibody fragment and including a monoclonal antibody.

[34] In addition, this invention includes a test kit for use in determining when a therapeutic or study agent should be discontinued for an individual being treated with a therapeutic or study agent; comprising one of the reagents above in a container suitable for contacting the said body fluid with instructions for interpreting the results. In a preferred embodiment this reagent comprises an antibody wherein said antibody specifically binds with the protein SAA.

[35] In other embodiments of this invention the step of determining the level of production of the gene expression product of the SAA gene in the said sample of tissue or body fluid is performed by determining the level of the mRNA expression product of the SAA gene in the said sample of tissue or body fluid, such as by means of: hybridization to a nucleotide array, Northern blot analysis, RT-PCR and real time quantitative PCR. [36] In some preferred embodiments the therapeutic or study agent is an epidermal growth factor receptor inhibitor (EGFRI), including but not limited to PKI166 or may be any oxidizing drug. DESCRIPTION OF THE PREFFERED EMBODIMENTS [37] It will be apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. In particular, although for clarity of disclosure, and not by way of limitation, the invention will be described with respect to the analysis of blood plasma samples, as one skilled in the art will appreciate, the assays and techniques described below can be applied to other biological fluid samples, e.g., cerebrospinal fluid (CSF), lymph, bile, plasma, whole blood, saliva, semen or urine; or tissue samples of any kind. The methods and compositions of the present invention are useful for the screening, diagnosis and prognosis of a living individual, but may also be used for post-mortem diagnosis in an individual.

[38] The present invention relates generally to the measurement of the messenger ribonucleic acid (mRNA) gene expression products and polypeptide or protein gene expression products of specific genes. The level of the expression products of these genes have been found to be correlated with liver and lung toxicity in patients treated with certain classes of drugs including, but not limited to, certain epidermal growth factor receptor inhibitors, such as PKI166. This invention is based in part on the discovery that a highly statistically significant correlation has been found between the pre-treatment level of the expression product of the gene SERPINA3 and the likelihood of the development, during drug treatment with a therapeutic or study agent, of hepatotoxicity and lung toxicity in patients treated with drugs that have such potential toxic effects including, but not limited to, epidermal growth factor receptor inhibitors, such as PKI166. The level of production of the gene expression product may be determined either by measuring the level of mRNA or protein/polypeptide expression product corresponding to the SERPINA3 gene. The polypeptide expression product of the SERPINA3 gene is the protease inhibitor, α-1 anti- chymotrypsin (AACT; SEQ ID NO:4). These levels may be measured in any way known to those of skill in the art.

[39] As used herein, the term "therapeutic agent" means a drug or other chemical compound administered to a patient in the hope of producing a therapeutic improvement in a recognized disease process.

[40] As used herein the term "aptamer" means a sequence of DNA or RNA that has selective protein binding characteristics. [41] As used herein, the term "study agent" means a drug or chemical compound administered to a patient with the primary purpose of determining the effect of that drug or compound on the patients disease process or on the patients general health or well being. [42] In addition, this invention is based, in part, on the discovery that a second protein, i.e., serum amyloid A (SAA; SEQ ID NO:4) is found to be low in all patients prior to treatment and was found to increase only in those individuals who developed dose-independent hepatotoxicity. SAA levels were found to increase earlier and more significantly than either AST or ALT and therefore provided a more accurate and earlier measure of adverse liver effects of therapy with drugs that have such potential toxic effects including but not limited to epidermal growth factor receptor inhibitors (EGFRIs) including, but not limited to, PKI166. [43] The levels of these two gene expression markers were not correlated with concomitant medication, type of cancer, pre-treatment liver function tests, age, sex or levels of the treatment drug.

[44] Thus, an aspect of the present invention are methods to predict the likelihood or probably that a particular individual will develop dose independent hepatotoxicity or lung toxicity when treated with a therapeutic or study agent that has such potential toxic effects including, but not limited to, an EGFRI, such as PKI166 or an oxidizing drug. [45] As used herein the term "oxidizing drug" shall mean a drug or chemical with the chemical properties of an oxidizing agent and includes but is not limited to EGFRI's, such as PKI166.

[46] Thus in one embodiment, this invention involves a method to determine the likelihood or probability that a particular patient will develop hepatotoxicity when treated with a given therapeutic agent including, but not limited to, an EGFRI, such as PKI166. This method also has utility for screening patients for inclusion or exclusion from a study or any kind of trial of a therapeutic agent for any reason, if that therapeutic or study agent could cause or is suspected of causing liver or lung toxicity

[47] This method involves the measurement of the level of the expression product of the SERPINA3 gene in the tissue or body fluids of an individual who is being considered for treatment with a therapeutic or study agent, such as an EGFRI including, but not limited to, PKI166. The level of expression in the said individual is compared with the average level of SERPINA3 gene expression in the tissues or body fluids in a number of control subjects. [48] These control subjects should be generally matched in terms of age, gender, health status with the individual being tested and the measurement of the level of the expression product of the SERPINA3 gene should be performed in the same manner and using the same type of tissue or body fluid. The number of control subjects employed should be 3-5 or in a more preferred embodiment 10-20 or more. The mean, average and standard deviation of the multiple levels so determined should be calculated by standard statistical means well- known to those of skill in the art.(See, Statistical Methods In Biology, by Norman T.J. Bailey, 3 rd Edition, Cambridge University Press 1995).

[49] The measured value from the individual or patient to be evaluated can then be compared to the average value so determined. If the level of the SERPINA3 gene expression product in the tissues or body fluids of the patient is significantly lower than the control average, this will indicate that the patient is in a high-risk category for the development of liver or lung toxicity during treatment with a therapeutic or study agent, such as an EGFRI or any agent with significant potential for liver or lung toxicity or marked oxidizing activity.

[50] The determination of when the difference in levels should be considered "significant" may be done in a variety of ways. In one embodiment, if the difference between the levels is more than 10% of the larger value this would be significant and would indicate that the individual is in a high-risk category for liver or lung toxicity. In another more preferred embodiment, if the individuals value is more than two standard deviations less that the average value, this would be significant and would indicate that the individual is in a high- risk category for liver or lung toxicity. In still other embodiments, if the difference between the two values is such that the probability of it occurring by chance alone is less than 5% (p<0.05) and the average value is the higher one, this would place the individual in a high- risk category.

[51] The determination of the level of the expression product of the SERPINA3 gene in the tissue or body fluids of an individual may be performed in a variety of ways. In one embodiment, this determination is made by measuring the level of the mRNA expression product of the SERPINA3 gene. Methods to measure the level of a specific mRNA are well- known in the art and include Northern blot analysis, reverse transcription PCR and real time quantitative PCR or by hybridization to a oligonucleotide array or microarray. In other more preferred embodiments, the determination of the level of expression of the SERPINA3 may be performed by determination of the level of the protein or polypeptide expression product of the gene in body fluids or tissue samples including but not limited to blood or serum. This protein or polypeptide expression product is the protease inhibitor alpha-1 anti-chymotrypsin (AACT). [52] The levels of this polypeptide gene expression product in body fluids or tissue samples may be determined by any means known in the art including Western blot analysis utilizing a labelled probe specific for the protein, use of so-called protein chips, immunoassays or ELISA techniques.. The said labelled probe may be an antibody or antibody fragment that recognizes the protein. These antibodies may be polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies. [53] In a preferred embodiment, the level of the protein in body fluids or tissues may be measured by means of mass spectrometric (MS) methods including, but not limited to, those techniques known in the art as matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF-MS) and surfaces enhanced for laser desorption/ionization, time-of-flight mass spectrometry (SELDI-TOF-MS). These techniques will be described in detail below.

[54] In another embodiment, this invention provides methods to detect and track the development of hepatotoxicity in an individual or patient undergoing treatment with a therapeutic agent that has the potential for causing liver toxicity, this includes, but is not limited to, an EGFRI, such as PKI166. These methods involve the measurement of the level of the protein Serum Amyloid A protein (SAA) in the body fluids or tissues of the individual or patient. The level of this protein remains low in patients who do not develop hepatotoxicity during treatment but increases in proportion to the development of liver damage or inflammation in response to a hepatotoxic therapeutic or study agent especially those known to be oxidizing including, including, but not limited to, an EGFRI, such as PKI166. [55] Thus, in one embodiment of this invention, methods are provided which involve the determination of the level of a gene expression product, i.e., the protein SAA in the body fluids or tissues of the individual or patient prior to initiation of the trial of the therapeutic or study agent and the determination of these protein levels one or more times during the course of treatment and at the end of treatment. If the levels of SAA increase significantly during treatment this indicates that the therapeutic or study agent is causing hepatotoxicity and appropriate modifications to the regimen of the therapeutic or study agent may be made accordingly, for example, the dose may be reduced or the drug may be stopped altogether and/or the individual removed from the study.

[56] The Detection of Nucleic Acids and Proteins as Markers. In a particular embodiment, the level of mRNA corresponding to the biomarkers of this invention can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated ribonucleic acid (RNA). For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from body fluids or tissue samples. See, e.g., Current Protocols in Molecular Biology, Ausubel et al., Ed., John Wiley & Sons, NY (1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well-known to those of skill in the art, such as, e.g., the single-step RNA isolation process of Chomczynski, U.S. Patent No. 4,843,155 (1989).

[57] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, e.g., a full-length complementary deoxyribonucleic acid (cDNA), or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic deoxyribonucleic acid (DNA) encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.

[58] In one format, the mRNA is immobilized on a solid surface and contacted with a probe, e.g., by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), e.g., in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.

[59] An alternative method for determining the level of mRNA corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, U.S. Patent No. 4,683,202 (1987); ligase chain reaction, Barany, Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 189-193 (1991); self-sustained sequence replication, Guatelli et al., Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 1874-1878 (1990); transcriptional amplification system, Kwoh et al., Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 1173-1177 (1989); Q-Beta Replicase, Lizardi et al, Bio/Technology, Vol. 6, p. 1197 (1988); rolling circle replication, Lizardi et al, U.S. Patent No. 5,854,033 (1988); or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art). These detection schemes are especially useful for the detection of the nucleic acid molecules if such molecules are present in very low numbers. [60] As used herein, the term "amplification primers" means a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and which contain a short region in between.

[61] In general, amplification primers are from about 10-30 nucleotides in length and flank a region from about 50-200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[62] For in situ methods, mRNA does not need to be isolated form the tissue sample cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.

[63] As an alternative to making determinations based on the absolute expression level of the marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes, such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a control sample, or between samples from different sources.

[64] Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of body fluid or tissue sample from controls , preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level.

[65] Detection of Polypeptide Markers. In another embodiment of the present invention, a polypeptide corresponding to a marker is detected. One agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide corresponding to a marker of the invention, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof, e.g., Fab or F(ab')₂ can be used. The term "labelled", with regard to the probe or antibody, is intended to encompass direct labelling of the probe or antibody by coupling, i.e., physically linking; a detectable substance to the probe or antibody, as well as indirect labelling of the probe or antibody by reactivity with another reagent that is directly labelled. Examples of indirect labelling include detection of a primary antibody using a fluorescently-labelled secondary antibody and end labelling of a DNA probe with biotin such that it can be detected with fluorescently-labelled streptavidin or by use of labelled aptamers. [66] Proteins from body fluids or tissue samples can be isolated using techniques that are well-known to those of skill in the art. The protein isolation methods employed can, e.g., be such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988). [67] A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA); radioimmunoasay (RIA), Western blot analysis and enzyme- linked immunosorbant assays (ELISAs). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether a sample of body fluid or a tissue sample contains a marker of the present invention.

[68] In one format, antibodies or antibody fragments, can be used in methods, such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite.

[69] One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from body fluids or tissue samples can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support, such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labelled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

[70] The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample, e.g., any body fluid including, but not limited to, serum, plasma, lymph, cystic fluid, urine, stool, CSF, acitic fluid or blood or a tissue sample. Such kits can be used to determine if a subject will be likely to experience hepatotoxicity or lung toxicity or is suffering from, or is at increased risk of, developing liver or lung toxicity. For example, the kit can comprise a labelled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample, e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide. Kits can also include instructions for interpreting the results obtained using the kit. [71] For antibody-based kits, the kit can comprise, e.g.: a) a first antibody, e.g., attached to a solid support, which binds to a polypeptide corresponding to a marker or the invention; and, optionally, b) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.

[72] For oligonucleotide-based kits, the kit can comprise, e.g.: a) an oligonucleotide, e.g., a detectably labelled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention; or b) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention.

[73] The kit can also comprise, e.g., a buffering agent, a preservative, or a protein- stabilizing agent. The kit can further comprise components necessary for detecting the detectable label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[74] Monitoring Clinical Trials. Monitoring the influence of agents, e.g., drug compounds; on the level of expression of a marker of the invention can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent to affect marker expression can be monitored in clinical trials of subjects receiving various trials of therapeutic agents. In a preferred embodiment, the present invention provides a method for monitoring the treatment of a subject with an agent, e.g., an agonist, antagonist and peptidomimetic; protein; peptide; nucleic acid; small molecule; or other drug candidate, comprising the steps of: (i) Obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) Detecting the level of expression of one or more selected markers of the invention in the pre-administration sample; (iii) Obtaining one or more post-administration samples from the subject; (iv) Detecting the level of expression of the marker(s) in the post-administration samples; (v) Comparing the level of expression of the marker(s) in the pre-administration sample with the level of expression of the marker(s) in the post-administration sample or samples; and (vi) Altering the administration of the agent to the subject accordingly. [75] For example, decreased or stopping the administration of the agent and/or removing the individual or patient from their study can be desirable if the markers demonstrate the likelihood of toxic effects occurring.

[76] The detection of the markers of the present invention can also be used to determine which individuals should be included or excluded from a drug study based on the likelihood that they will experience toxic effects from the administration of the study drug. [77] Detection and Measurement of Biomarkers. Expression of the protein encoded by the gene(s) disclosed herein can be detected by a probe which is detectably-labelled, or which can be subsequently labelled. In some embodiments, the probe is an antibody that recognizes the expressed protein.

[78] As used herein, the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional antibody fragments sufficient for binding of the antibody fragment to the protein. [79] For the production of antibodies to a protein encoded by one of the disclosed genes, various host animals may be immunized by injection with the polypeptide, or a portion thereof. Such host animals may include, but are not limited to, rabbits, mice and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species including, but not limited to, Freund's (complete and incomplete) mineral gels, such as aluminium hydroxide; surface active substances, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet haemocyanin and dinitrophenol; and potentially useful human adjuvants, such as bacille Camette-Guerin (BCG) and Corynebacterium parvum.

[80] Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals, such as those described above, may be immunized by injection with the encoded protein, or a portion thereof, supplemented with adjuvants as also described above. [81] Monoclonal antibodies (mAbs), which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, Nature, Vol. 256, pp. 495-497 (1975); and U.S. Patent No.4,376,110. The human B-cell hybridoma technique of Kosbor et al., Immunol. Today, Vol. 4, No. 72 (1983); Cole et al, Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 2026-2030 (1983); and the EBV-hybridoma technique, Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titres of mAbs in vivo makes this the presently preferred method of production.

[82] In addition, techniques developed for the production of "chimeric antibodies", Morrison et al, Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 6851-6855 (1984); Neuberger et al, Nature, Vol. 312, pp. 604-608 (1984); Takeda et al, Nature, Vol. 314, pp.452-454 (1985), by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived form a murine mAb and a human immunoglobulin constant region. [83] Alternatively, techniques described for the production of single-chain antibodies, U.S. Patent No. 4,946,778; Bird, Science, Vol. 242, No. 4877, pp. 423-426 (1988); Huston et a/., Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 5879-5883 (1988); and Ward et al, Nature, Vol. 334, pp. 544-546 (1989), can be adapted to produce differentially-expressed gene single- chain antibodies. Single-chain antibodies are formed by linking the heavy- and light-chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide. [84] More preferably, techniques useful for the production of "humanized antibodies" can be adapted to produce antibodies to the proteins, fragments or derivatives thereof. Such techniques are disclosed in U.S. Patent Nos. 5,932,448; 5,693,762; 5,693,761 ; 5,585,089; 5,530,101 ; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,661,016 and 5,770,429. [85] Antibody fragments, which recognize specific epitopes, may be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed, Huse et al, Science, Vol. 246, No. 4935, pp. 1275-1281 (1989), to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[86] The extent to which the known proteins are expressed in the sample is then determined by immunoassay methods that utilize the antibodies described above. Such immunoassay methods include, but are not limited to, dot blotting, western blotting, competitive and non-competitive protein binding assays, ELISAs, immunohistochemistry, fluorescence activated cell sorting (FACS) and others commonly-used and widely-described in scientific and patent literature, and many employed commercially. [87] In some embodiments, for ease of detection, the sandwich ELISA may be used, of which a number of variations exist, all of which are intended to be encompassed by the present invention. For example, in a typical forward assay, unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule after a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex. At this point, a second antibody, labelled with a reporter molecule capable of inducing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen- labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. [88] Variations on the forward assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and added to the unlabeled surface bound antibody. These techniques are well-known to those skilled in the art, and the possibility of minor variations will be readily apparent. As used herein, "sandwich assay" is intended to encompass all variations on the basic two-site technique. For the immunoassays of the present invention, the only limiting factor is that the labelled antibody must be an antibody that is specific for the protein expressed by the gene of interest.

[89] The most commonly used reporter molecules in this type of assay are either enzymes, fluorophore- or radionuclide-containing molecules. In the case of an enzyme immunoassay an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different ligation techniques exist, which are well-known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1 ,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. A solution containing the appropriate substrate is then added to the tertiary complex. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an evaluation of the amount of protein which is present in the serum sample.

[90] Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic colour visually detectable with a light microscope. Immunofluorescence and EIA techniques are both very well-established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.

[91] Measurement of the translational state may also be performed according to several additional methods. For example, whole genome monitoring of protein, i.e., the "proteome", Goffeau et al, supra, can be carried out by constructing a microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome. Preferably, antibodies are present for a substantial fraction of the encoded proteins, or at least for those proteins relevant to testing or confirming a biological network model of interest. Methods for making monoclonal antibodies are well-known. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)., which is incorporated in its entirety for all purposes. In a one preferred embodiment, monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell. With such an antibody array, proteins from the cell are contacted to the array, and their binding is assayed with assays known in the art. [92] Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well-known in the art and typically involves isoelectric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. See, e.g., Hames et al, Gel Electrophoresis of Proteins: A Practical Approach (IRL Press, NY, 1990); Shevchenko et al, Proc Natl Acad Sci U S A, Vol. 93, No. 4, pp. 1440-1445 (1996); Sagliocco et al, Yeast, Vol. 12, No. 15, pp. 1519-1533 (1996); and Lander, Science, Vol. 274, No. 5287, pp. 536-539 (1996). The resulting electropherograms can be analyzed by numerous techniques, including MS techniques, western blotting and immunoblot analysis using polyclonal and monoclonal antibodies, and internal and Λ/-terminal micro-sequencing. Using these techniques, it is possible to identify a substantial fraction of all the proteins produced under given physiological conditions, including in cells, e.g., in yeast, exposed to a drug, or in cells modified by, e.g., deletion or over-expression of a specific gene.

[93] Embodiments Based on Other Aspects of the Biological State. It will be apparent to those of skill in the art that in the use of methods of this invention that the activities of proteins that are the markers of the present invention can also be measured, embodiments of this invention can be based on such measurements. Activity measurements can be performed by any functional, biochemical or physical means appropriate to the particular activity being characterized. Where the activity involves a chemical transformation such as enzymatic activity, the protein can be contacted with the natural substrates, and the rate of transformation measured. Where the activity involves association in multimeric units, e.g., association of an activated DNA binding complex with DNA, the amount of associated protein or secondary consequences of the association, such as amounts of mRNA transcribed, can be measured.

[94] Also, where only a functional activity is known performance of the function can be observed. However known and measured, the changes in protein activities may form the response data analyzed by the foregoing methods of this invention. In alternative and non- limiting embodiments, response data may be formed of mixed aspects of the biological state of a cell. Response data can be constructed from, e.g., changes in certain mRNA abundances, changes in certain protein abundances and changes in certain protein activities.

[95] Detection Methods. Methods of detecting the level of expression of mRNA are well- known in the art and include, but are not limited to, northern blotting, reverse transcription PCR, real time quantitative PCR and other hybridization methods such as hybridization to a oligonucleotide array.

[96] A particularly useful method for detecting the level of mRNA transcripts obtained from a plurality of the disclosed genes involves hybridization of labelled mRNA to an ordered array of oligonucleotides. Such a method allows the level of transcription of a plurality of these genes to be determined simultaneously to generate gene expression profiles or patterns. The gene expression profile derived from the sample obtained from the subject can, in another embodiment, be compared with the gene expression profile derived form the sample obtained from the disease-free subject, and thereby determine whether the subject has or is at risk of developing hepatotoxicity.

[97] In further preferred embodiments, the levels of the gene expression products (proteins) can be monitored in various body fluids including, but not limited to, blood, plasma, serum, lymph, cerebro-spinal fluid (CSF), cystic fluid, ascites, urine, saliva, stool and bile. This expression product level can be used as surrogate markers.

[98] Proteomics. Proteins that are secreted into body fluids or tissues can be analyzed to identify those proteins that are useful as biomarkers and may be of value in the methods of this invention. Supernatants can be isolated and MWT-CO filters can be used to simplify the mixture of proteins. The proteins can then be digested with trypsin. The tryptic peptides may then be loaded onto a micro capillary HPLC column where they are separated, and eluted directly into an ion trap mass spectrometer, through a custom-made electrospray ionization source. Throughout the gradient, sequence data can be acquired through fragmentation of the four most intense ions (peptides) that elute off the column, while dynamically excluding those that have already been fragmented. In this way, the sequence data from multiple scans can be obtained, corresponding to approximately 50-200 different proteins in the sample. These data are searched against databases using correlation analysis tools, such as MS-Tag, to identify the_proteins in the supernatants. [99] MASLDI-TOF-MS. In some preferred embodiments, the detection of specific proteins or poly peptide gene expression products in body fluids or tissue samples would be performed by means of mass spectrometry (MS), especially matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MASLDI-TOF-MS). These techniques have been used to analyze macromolecules, such as proteins or biomolecules and utilize sample probe surface chemistries that enable the selective capture and desorption of analytes, including intact macromolecules, directly from the probe surface into the gas (vapour phase), and in the most preferred embodiments without added chemical matrix. Generally, analysis by MS involves the vaporization and ionization of a small sample of material, using a high energy source, such as a laser, including a laser beam. The material is vaporized from the surface of a probe tip into the gas or vapour phase by the laser beam, and, in the process, some of the individual molecules are ionized by the gain of a proton. The positively charged ionized molecules are then accelerated through a short high voltage field and let fly (drift) into a high vacuum chamber, at the far end of which they strike a sensitive detector surface. Since the time-of-flight is a function of the mass of the ionized molecule, the elapsed time between ionization and impact can be used to determine the molecule's mass which, in turn, can be used to identify the presence or absence of known molecules of specific mass.

[100] In some embodiments of this technique, this procedure which presents proteins or other large biomolecules on a probe tip for laser desorption/ionization TOF-MS relies on the preparation of a crystalline solid mixture of the protein or other analyte molecule in a large molar excess of acidic matrix material deposited on the bare surface of a metallic probe tip (the sample probe tip typically is metallic, either stainless steel, nickel plated material or platinum). Embedding the analyte in such a matrix is intended to prevent the destruction of analyte molecules by the laser beam. The laser beam strikes the solid mixture on the probe tip and its energy is used to vaporize a small portion of the matrix material along with some of the embedded analyte molecules. Without the matrix, the analyte molecules are easily fragmented by the laser energy, so that the mass, and identity, of the original macromolecule is very difficult or impossible to determine.

[101] In other embodiments, UV laser for the desorption process is used followed by TOF-MS. This allows proteins of relatively high molecular mass to be deposited on the probe tip in the presence of a very large molar excess of an acidic, UV absorbing chemical matrix, such as nicotinic acid. Under these circumstances, i.e., with the addition of the chemical matrix, high molecular mass biopolymers, such as proteins and nucleic acids could be desorbed in the intact state. This new technique is called MALDI-TOF-MS. [102] In other embodiments a variety of other techniques for marker detection using mass spectroscopy may be used. See Bordeaux Mass Spectrometry Conference Report, Hillenkamp, Ed., pp. 354-362 (1988); Bordeaux Mass Spectrometry Conference Report, Karas and Hillenkamp, Eds., pp. 416-417 (1988); Karas and Hillenkamp, Anal Chem, Vol. 60, pp. 2299-2301 (1988); and Karas et al, Biomed Environ Mass Spectrum, Vol. 18, pp. 841-843 (1989). The use of laser beams in TOF-MS is shown, e.g., in U.S. Patent Nos. 4,694,167; 4,686,366, 4,295,046 and 5,045,694, all incorporated by reference in their entirety herein.

[103] Other MS techniques allow the successful volatilization of high molecular weight biopolymers, without fragmentation, and have enabled a wide variety of biological macromolecules to be analyzed by mass spectrometry.

[104] Sudaces Enhanced for Laser Desorption/ionization (SELDI). In a preferred embodiment of the present invention other techniques are used which employ new MS probe element compositions with surfaces that allow the probe element to actively participate in the capture and docking of specific analytes, described as Affinity Mass Spectrometry (AMS). Several types of new MS probe elements have been designed (see Hutchens and Yip, Rapid Commun Mass Spectrom, Vol. 7, pp. 576- 580 (1993)) with Surfaces Enhanced for Affinity Capture (SEAC). SEAC probe elements have been used successfully to retrieve and tether different classes of biopolymers, particularly proteins, by exploiting what is known about protein surface structures and biospecific molecular recognition. [105] The immobilized affinity capture devices on the MS probe element surface, i.e., SEAC, determines the location and affinity (specificity) of the analyte for the probe surface, therefore the subsequent analytical AMS process is much more efficient for several reasons. [106] First, the location of analyte on the probe element surface is pre-determined. Thus, the subsequent desorption is no longer dependent on a random search of the probe surface matrix field with the incident laser beam. [107] Second, analyte detection sensitivity (and dynamic range) is increased because molecular ionization suppression effects often observed with complex mixtures are eliminated.

[108] Third, the tethered analyte that is not actually consumed by the initial laser-induced desorption process remains available for subsequent analyses. If exogenous matrix was used to promote analyte desorption, it is removed, in most cases, without loss of the tethered analyte. The remaining analyte can then be chemically and/or enzymatically modified directly in situ, i.e., while still on the probe element. When analyzed again by MS to determine differences in mass, specific structural details are revealed. The entire process of analysis/modification can be repeated many times to derive structural information while consuming only very small quantities of analyte (sometimes only a few femtomoles or less). The demonstrations of protein structure analysis based on AMS have included both N- and C-terminal sequence analyses and verification of several types of sequence-specific posttranslational modifications including phosphorylation and dephosphorylation; glycosylation; cysteine residue reactivity; site-specific chemical modifications, e.g., histidine residues; and ligand binding.

[109] In a most preferred embodiment of this invention the method of detection to be used with the methods of this invention uses a general category of probe elements, i.e., sample presenting means with surfaces enhanced for laser desorption/ionization (SELDI). See SELDI patents U.S. Patent Nos. 5,719,060; 5,894,063; 6,020,208; 6,027,942, 6,124,137 and U.S. Patent application No. U.S. 2003/0003465, all incorporated by reference herein, in their entirety and for all purposes.

[110] Within this general category there are three (3) separate subcategories. [111] Category 1 : Surfaces Enhanced for Neat Desorption (SEND), where the probe element surfaces, i.e., sample presenting means, are designed to contain Energy Absorbing Molecules (EAM) instead of "matrix" to facilitate desorption/ionizations of analytes added directly (neat) to the surface. Note: Category 1 (SEND) is used alone or in combination with the following Category 2.

[112] Category 2: SEAC, where the probe element surfaces, i.e., sample presenting means, are designed to contain chemically defined and/or biologically defined affinity capture devices to facilitate either the specific or non-specific attachment or adsorption (so-called docking or tethering) of analytes to the probe surface, by a variety of mechanisms (mostly non-covalent). Note: Category 2 (SEAC) is used with added matrix or it is used in combination with Category 1 (SEND) without added matrix. Thus, the combination of SEND and SEAC actually represents a distinctive category.

[113] Category 3: Surfaces Enhanced for Photolabile Attachment and Release (SEPAR), where the probe element surfaces, i.e., sample presenting means, are designed/modified to contain one or more types of chemically defined cross-linking molecules to serve as covalent docking devices. These Photolabile Attachment Molecules (PAM) are bivalent or multivalent in character, that is, one side is first reacted so as to permanently attach the PAM to the probe element surface of the sample presenting means, then the other reactive side(s) of the PAM is ready to be reacted with the analyte when the analyte makes contact with the PAM- derivatised probe surface. Such surfaces, i.e., sample presenting means, allow for very strong, i.e., stable or covalent; analyte attachment or adsorption, i.e., docking or tethering; processes that are covalent but reversible upon irradiation, i.e., photolabile. Such surfaces represent platforms for the laser-dependent desorption of analytes that are to be chemically and/or enzymatically modified in situ, i.e., directly on the probe tip, for the purpose of structure elucidation. Only those analytes on the probe surface that are actually irradiated (small percentage of total) is desorbed. The remainder of the tethered analytes remain covalently bound and is modified without loss due to some inadvertent uncoupling from the surface. Note: Category 3 (SEPAR) is characterized by analyte attachment processes that are reversible upon exposure to light. However, the light-dependant reversal of the analyte surface attachment bond(s) does not necessarily enable analyte desorption into the gas phase per se.

[114] In other words, the molecules responsible for the photolabile attachment of the analytes to the probe surface are not necessarily the same as the Energy Absorbing Molecules (EAM) described for SEND. Some embodiments include some hybrid EAM/PAM chemicals that have dual functionality with respect to SEND and SEPAR. That is, some EAM molecules presently used for SEND can be modified to act as mediators of both the SEND and SEPAR processes.

[115] In addition, some hybrid affinity capture/PAM chemicals have dual functionality with respect to SEAC and SEPAR are used and in some embodiments affinity capture devices, particularly those that are biologically defined, may be modified to act as mediators of both the SEAC and SEPAR processes.

[116] Thus, this method involves a sample presenting means, i.e., probe element surface, with surface-associated (or surface-bound) molecules to promote the attachment (tethering or anchoring) and subsequent detachment of tethered analyte molecules in a light- dependent manner, wherein the said surface molecule(s) are selected from the group consisting of photoactive (photolabile) molecules that participate in the binding (docking, tethering or cross-linking) of the analyte molecules to the sample presenting means (by covalent attachment mechanisms or otherwise).

[117] The chemical specificity(ies) determining the type and number of said photolabile molecule attachment points between the SEPAR sample presenting means, i.e., probe element surface, and the analyte, e.g., protein, may involve any one or more of a number of different residues or chemical structures in the analyte, e.g., His, Lys, Arg, Tyr, Phe and Cys residues in the case of proteins and peptides. In other words, in the case of proteins and peptides, the SEPAR sample presenting means may include probe surfaces modified with several different types of photolabile attachment molecules to secure the analyte(s) with a plurality of different types of attachment points.

[118] The wavelength of light or light intensity (or incident angle) required to break the photolabile attachment(s) between the analyte and the probe element surface may be the same or different from the wavelength of light or light intensity (or incident angle) required to promote the desorption of the analyte from the stationary phase into the gas or vapour phase.

[119] The photolabile attachment of the analyte(s) to the probe element surface, i.e., sample presenting means, particularly biopolymers, such as peptides, proteins, RNAs, DNAs and carbohydrates, may involve multiple points of attachment between the probe surface and the analyte macromolecule. Once the biopolymer is attached via multiple points of attachment, different points in the backbone of the biopolymer may be deliberately cut or fragmented by chemical and/or enzymatic means so that many of the resulting fragments are now separate and distinct analytes, each one still attached (tethered) to the probe surface by one or more photolabile bonds, to be desorbed into the gas phase in parallel for simultaneous mass analyses with a TOF mass analyzer. This process enables biopolymer (protein, peptides, RNA, DNA, carbohydrate) sequence determinations to be made. [120] Thus one preferred embodiment, for the detection of the biomarkers of the present invention, is an apparatus for measuring the mass of an analyte molecule of an analyte sample by means of MS, said apparatus comprising: a) a spectrometer tube; b) vacuum means for applying a vacuum to the interior of said tube; c) electrical potential means within the tube for applying an accelerating electrical potential to desorbed analyte molecules from said analyte sample; d) sample presenting means irremovably insertable into said spectrometer tube, for presenting said analyte sample in association with surface associated molecule for promoting desorption and ionization of said analyte molecules, wherein said surface molecule is selected from the group consisting of energy absorbing molecule, affinity capture device, photolabile attachment molecule and combination thereof; e) an analyte sample deposited on said sample presenting means in association with said surface associated molecules, whereby at least a portion of said analyte molecules not consumed in said MS analysis will remain accessible for subsequent chemical, biological or physical analytical procedures; f) laser beam means for producing a laser beam directed to said analyte sample for imparting sufficient energy to desorb and ionize a portion of said analyte molecules from said analyte sample; and g) detector means associated with said spectrometer tube for detecting the impact of accelerated ionized analyte molecules thereon.

Immobilization of a Polypeptide to a Solid Support

[121] For MS analyses, a target polypeptide or other polypeptide of interest can be conjugated and immobilized to a solid support in order to facilitate manipulation of the polypeptide. Such supports are well-known to those of skill in the art, and include any matrix used as a solid support for linking proteins. The support is selected to be impervious to the conditions of MS analyses. Supports, which can have a flat surface or a surface with structures include, but are not limited to, beads, such as silica gel beads, controlled pore glass beads, magnetic beads and Dynabeads; Wang resin; Merrifield resin, SEPHADEX/SEPHAROSE™ beads or cellulose beads; capillaries; flat supports, such as glass fibre filters, glass surfaces and metal surfaces, including steel, gold silver, aluminium, silicon and copper; plastic materials, including multiwell plates or membranes (formed, e.g., of polyethylene, polypropylene, polyamide or polyvinylidene difluoride); wafers; combs; pins or needles, including arrays of pins suitable for combinatorial synthesis or analysis; beads, in an array of pits; wells, particularly nanoliter wells, in flat surfaces, including wafers, such as silicon wafers; and wafers with pits, with or without filter bottoms. A solid support is appropriately functionalized for conjugation of the polypeptide and can be of any suitable shape appropriate for the support.

[122] A solid support, such as a bead, can be functionalized for the immobilization of polypeptides, and the bead can be further associated with a solid support, if desired. Where a bead is to be conjugated to a second solid support, polypeptides can be immobilized on the functionalized support before, during or after the bead is conjugated to the second support.

[123] A polypeptide of interest can be conjugated directly to a solid support or can be conjugated indirectly through a functional group present either on the support, or a linker attached to the support or the polypeptide or both. For example, a polypeptide can be immobilized to a solid support due to a hydrophobic, hydrophilic or ionic interaction between the support and the polypeptide. Although such a method can be useful for certain manipulations, such as for conditioning of the polypeptide prior to MS, such a direct interaction is limited in that the orientation of the polypeptide is not known and can be random based on the position of the interacting amino acids, e.g., hydrophobic amino acids, in the polypeptide. Thus, a polypeptide generally is immobilized in a defined orientation by conjugation through a functional group on either the solid support or the polypeptide or both. [124] A polypeptide of interest can be modified by adding an appropriate functional group to the carboxyl terminus or amino terminus of the polypeptide, or to an amino acid in the peptide, e.g., to a reactive side chain, or to the peptide backbone. It should be recognized, however, that a naturally-occurring amino acid normally present in the polypeptide also can contain a functional group suitable for conjugating the polypeptide to the solid support. For example, a cysteine residue present in the polypeptide can be used to conjugate the polypeptide to a support containing a sulfhydryl group, e.g., a support having cysteine residues attached thereto, through a disulfide linkage. Other bonds that can be formed between two amino acids, include, e.g., monosulfide bonds between two lanthionine residues, which are non-naturally-occurring amino acids that can be incorporated into a polypeptide; a lactam bond formed by a transamidation reaction between the side chains of an acidic amino acid and a basic amino acid, such as between the y-carboxyl group of Glu (or alpha carboxyl group of Asp) and the amino group of Lys; or a lactone bond produced, e.g., by a crosslink between the hydroxy group of Ser and the carboxyl group of Glu (or alpha carboxyl group of Asp). Thus, a solid support can be modified to contain a desired amino acid residue, e.g., a Glu residue, and a polypeptide having a Ser residue, particularly a Ser residue at the carboxyl terminus or amino terminus, can be conjugated to the solid support through the formation of a lactone bond. It should be recognized, however, that the support need not be modified to contain the particular amino acid, e.g., Glu, where it is desired to form a lactone-like bond with a Ser in the polypeptide, but can be modified, instead, to contain an accessible carboxyl group, thus providing a function corresponding to the alpha carboxyl group of Glu. [125] A polypeptide of interest also can be modified to facilitate conjugation to a solid support, e.g., by incorporating a chemical or physical moiety at an appropriate position in the polypeptide, generally the C-terminus or N-terminus. The artisan will recognize, however, that such a modification, e.g., the incorporation of a biotin moiety, can affect the ability of a particular reagent to interact specifically with the polypeptide and, accordingly, will consider this factor, if relevant, in selecting how best to modify a polypeptide of interest. [126] In one aspect of the processes provided herein, a polypeptide of interest can be covalently conjugated to a solid support and the immobilized polypeptide can be used to capture a target polypeptide, which binds to the immobilized polypeptide. The target polypeptide then can be released from immobilized polypeptide by ionisation or volatisation for MS, whereas the covalently conjugated polypeptide remains bound to the support. [127] Accordingly, a method to determine the identity of polypeptides that interact specifically with a polypeptide of interest is provided. For example, such a process can be used to determine the identity of target polypeptides obtained from one or more biological samples that interact specifically with the immobilized polypeptide of interest. Such a process also can be used, e.g., to determine the identity of binding proteins such as antibodies that bind to the immobilized polypeptide antigen of interest, or receptors that bind to an immobilized polypeptide ligand of interest, or the like. Such a process can be useful, e.g., for screening a combinatorial library of modified target polypeptides, such as modified antibodies, antigens, receptors, hormones or other polypeptides to determine the identity of those target polypeptides that interact specifically with the immobilized polypeptide. [128] In one aspect of the processes provided herein, a polypeptide of interest can be covalently conjugated to a solid support and the immobilized polypeptide can be used to capture a target polypeptide, which binds to the immobilized polypeptide. The target polypeptide then can be released from immobilized polypeptide by ionisation or volatisation for MS, whereas the covalently conjugated polypeptide remains bound to the support. [129] Accordingly, in some embodiments of this invention a process is provided to determine the identity of polypeptides that interact specifically with a polypeptide of interest. For example, such a process can be used to determine the identity of target polypeptides obtained from one or more biological samples that interact specifically with the immobilized polypeptide of interest. Such a process also can be used, e.g., to determine the identity of binding proteins, such as antibodies that bind to the immobilized polypeptide antigen of interest, or receptors that bind to an immobilized polypeptide ligand of interest, or the like. Such a process can be useful, e.g., for screening a combinatorial library of modified target polypeptides, such as modified antibodies, antigens, receptors, hormones or other polypeptides to determine the identity of those target polypeptides that interact specifically with the immobilized polypeptide.

[130] A polypeptide of interest can be conjugated to a solid support, which can be selected based on advantages that can be provided. Conjugation of a polypeptide to a support, e.g., provides the advantage that a support has a relatively large surface area for immobilization of polypeptides. A support, such as a bead, can have any three dimensional structure, including a surface to which a polypeptide, functional group or other molecule can be attached. If desired, a support, such as a bead, can have the additional characteristic that it can be conjugated further to a different solid support, e.g., to the walls of a capillary tube. A support useful for the disclosed processes or kits generally has a size in the range of about 1 micrometer to about 1000 micrometers in diameter; can be made of any insoluble or solid material, as disclosed above; and can also be a swellable bead, e.g., a polymeric bead, such as Wang resin, or a non-swellable bead, such as a controlled pore glass. [131] A solid surface also can be modified to facilitate conjugation of a polypeptide of interest. A thiol-reactive functionality is particularly useful for conjugating a polypeptide to a solid support. A thiol-reactive functionality is a chemical group that can rapidly react with a nucleophilic thiol moiety to produce a covalent bond, e.g., a disulfide bond or a thioether bond. In general, thiol groups are good nucleophiles and, therefore, thiol-reactive functionalities generally are reactive electrophiles. A variety of thiol-reactive functionalities are known in the art, including, e.g., haloacetyls, such as iodoacetyl; diazoketones; epoxy ketones, alpha;- and beta;-unsaturated carbonyls, such as alpha;-enones and beta;-enones; and other reactive Michael acceptors, such as maleimide; acid halides; benzyl halides; and the like. A free thiol group of a disulfide, e.g., can react with a free thiol group by disulfide bond formation, including by disulfide exchange. Reaction of a thiol group can be temporarily prevented by blocking with an appropriate protecting group, as is conventional in the art. See Greene and Wuts, Protective Groups in Organic Synthesis, 2^nd Edition, John Wiley & Sons (1991).

[132] Reducing agents that are useful for reducing a polypeptide containing a disulfide bond include tris-(2-carboxyethyl)phosphine (TCEP), which generally is used in a concentration of about 1-100 mM, usually about 10 mM, and is reacted at a pH of about 3-6, usually at a pH of about 4.5, a temperature of about 20-45°C, usually about 37°C, for about 1-10 hours, usually about 5 hours; dithiothreitol, which generally is used in a concentration of about 25-100 mM, and is reacted at a pH of about 6-10, usually at a pH of about 8, a temperature of about 25-45°C, usually about 37°C, for about 1-10 hours, usually about 5 hours. TCE provides an advantage in that it is reactive at a low pH, which effectively protonates thiols, thus suppressing nucleophilic reactions of thiols and resulting in fewer side reactions than with other disulfide reducing agents.

[133] A thiol-reactive functionality, such as 3-mercaptopropyltriethoxysilane can be used to functionalize a silicon surface with thiol groups. The amino functionalized silicon surface then can be reacted with a heterobifunctional reagent, such as Λ/-succinimidyl (4-iodacetyl) aminobenzoate (SIAB) (Pierce; Rockford III.). If desired, the thiol groups can be blocked with a photocleavable protecting group, which then can be selectively cleaved, e.g., by photolithography, to provide portions of a surface activated for immobilization of a polypeptide of interest. Photocleavable protecting groups are known in the art (see, e.g., published International PCT Application No. WO 92/10092; and McCray et al, Ann Rev Biophys Biophys Chem, Vol. 18, pp. 239-270 (1989)) and can be selectively de-blocked by irradiation of selected areas of the surface using, e.g., a photolithography mask. [134] Linkers. As noted herein, the polypeptide can be linked either directly to the support or via a linking moiety or moieties. Any linkers known to those of skill in the art to be suitable for linking peptides or amino acids to supports, either directly or via a spacer, may be used. Linkers, include, Rink amide linkers (see, e.g., Rink, Tetrahedron Lett, Vol. 28, p. 3787 (1976); trityl chloride linkers (see, e.g., Leznoff, Ace Chem Res, Vol. 11 , p. 327 (1978); and Merrifield linkers. See, e.g., Bodansky et al, Peptide Synthesis, Academic Press, 2^nd Edition, NY (1976).

[135] For example, trityl linkers are known. See, e.g., U.S. Patent Nos. 5,410,068 and 5,612,474. Amino trityl linkers are also known. See, e.g., U.S. Patent No. 5,198,531. Linkers that are suitable for chemically linking peptides to supports, include disulfide bonds, thioether bonds, hindered disulfide bonds and covalent bonds between free reactive groups, such as amine and thiol groups. These bonds can be produced using heterobifunctional reagents to produce reactive thiol groups on one or both of the polypeptides and then reacting the thiol groups on one polypeptide with reactive thiol groups or amine groups on the other.

[136] Other linkers include, acid cleavable linkers, such as /./s-maleimideothoxy propane, acid labile-transferrin conjugates and adipic acid diihydrazide, that would be cleaved in more acidic intracellular compartments; photocleavable cross linkers that are cleaved by visible or UV light, RNA linkers that are cleavable by ribozymes and other RNA enzymes, and linkers, such as the various domains, such as CHi, CH₂ and CH₃, from the constant region of human lgG1. See, Batra et al., Mol Immunol, Vol. 30, pp. 379-396 (1993). [137] Any linker known to one skilled in the art for immobilizing a polypeptide to a solid support can be used in a process as disclosed herein. Combinations of any linkers are also contemplated herein. For example, a linker that is cleavable under MS conditions, such as a silyl linkage or photocleavable linkage, can be combined with a linker, such as an avidin biotin linkage, that is not cleaved under these conditions, but may be cleaved under other conditions.

[138] A polypeptide of interest can be attached directly to a support via a linker. For example, the polypeptide can be conjugated to a support, such as a bead, through means of a variable spacer. In addition, the conjugation can be directly cleavable, e.g., through a photocleavable linkage, such as a streptavidin or avidin to biotin interaction, which can be cleaved by a laser as occurs for MS, or indirectly through a photocleavable linker (see U.S. Patent No. 5,643,722) or an acid labile linker, heat sensitive linker, enzymatically cleavable linker or other such linker.

[139] A linker can provide a reversible linkage such that it is cleaved under the conditions of MS. Such a linker can be, e.g., a photo-cleavable bond, such as a charge transfer complex or a labile bond formed between relatively stable organic radicals. A linker (L) on a polypeptide can form a linkage, which generally is a temporary linkage, with a second functional group (U) on the solid support.

[140] Furthermore, where the polypeptide of interest has a net negative charge, or is conditioned to have such a charge, the linkage can be formed with U being, e.g., a quaternary ammonium group. In this case, the surface of the solid support carries a negative charge that repels the negatively charged polypeptide, thereby facilitating desorption of the polypeptide for MS analysis. Desorption can occur due to the heat created by the laser pulse or, where L' is a chromophore, by specific absorption of laser energy that is in resonance with the chromophore.

[141] A linkage (L-U) can be, e.g., a disulfide bond, which is chemically cleavable by mercaptoethanol or dithioerythrol; a biotin/streptavidin linkage, which can be photocleavable; a heterobifunctional derivative of a trityl ether group, which can be cleaved by exposure to acidic conditions or under conditions of MS (see Koster et al, Tetrahedron Lett, Vol. 31 , p. 7095 (1990)); a levulinyl-mediated linkage, which can be cleaved under almost neutral conditions with a hydrazinium/acetate buffer; an arginine-arginine or a lysine-lysine bond, either of which can be cleaved by an endopeptidase, such as trypsin; a pyrophosphate bond, which can be cleaved by a pyrophosphatase; or a ribonucleotide bond, which can be cleaved using a ribonuclease or by exposure to alkali condition.

[142] The functionalities, L and L', can also form a charge transfer complex, thereby forming a temporary L-U linkage. Since the "charge-transfer band" can be determined by UV/vis spectrometry (see Foster, Organic Charge Transfer Complexes, Academic Press (1969)), the laser energy can be tuned to the corresponding energy of the charge-transfer wavelength and specific desorption from the solid support can be initiated. It will be recognized that several combinations of L and L' can serve this purpose and that the donor functionality can be on the solid support or can be coupled to the polypeptide to be detected or vice versa.

[143] A reversible L-L' linkage also can be generated by homolytically forming relatively stable radicals. Under the influence of the laser pulse, desorption, as well as ionization, can take place at the radical position. Various organic radicals can be selected such that, in relation to the dissociation energy needed to homolytically cleave the bond between the radicals, a corresponding laser wavelength can be selected. See Reactive Molecules, Wentrup, Ed., John Wiley & Sons (1984).

[144] Other linkers include those that can be incorporated into fusion proteins and expressed in a host cell. Such linkers may be selected amino acids, enzyme substrates or any suitable peptide. The linker may be made, e.g., by appropriate selection of primers when isolating the nucleic acid. Alternatively, they may be added by post-translational modification of the protein of interest.

[145] In particular, selectively cleavable linkers, including photocleavable linkers, acid cleavable linkers, acid-labile linkers and heat sensitive linkers are useful. Acid cleavable linkers include, e.g., bismaleimideothoxy propane, adipic acid dihydrazide linkers (see Fattom et a/., Infect Immun, Vol. 60, pp. 584-589 (1992)), and acid labile transferrin conjugates that contain a sufficient portion of transferrin to permit entry into the intracellular transferrin cycling pathway. See Welhoner et al., J Biol Chem, Vol. 266, pp. 4309-4314 (1991).

[146] The capture of a polypeptide may be through the amino-terminus of the peptide. Herer, the peptide may be, for example, captured onto a surface of a support through the use of a diisopropylysilyl diether group. Other silyl diether groups including, but not limited to, dialkylsilyl, diarylsilyl and alkylarylsilyl, may also be used. Reaction of a hydroxylated support surface with duiisopropylsilyl dichloride and a hydroxyester provides the starting surface-bound diisopropylysilyl diether ester. [147] The hydroxylated support surface may be prepared by methods that are well-known to those of skill in the art. For example, Λ/-succinimidyl(4-iodacetyl) aminobenzoate (SIAB). Other agents as linkers include, but are not limited to, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-S-acetyl-thioacetate (SATA), Λ/-succinimidyl-3-(2-pyridyldithiol propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-hydrazinonicotimide (HYNIC) may also be used. For further examples of cross-linking reagents, see, e.g., Chemistry of Protein Conjugation and Cross-Linking, Wong, Ed., CRC Press (1991) and Bioconiugate Techniques, Hermanson, Ed., Academic Press (1995). Hydroxyesters that may be used include, but are not limited to, hydroxyacetate (glycolate), alpha, beta and gamma;-hydroxylakanotates, gamma;-hydroxy(polyethyleneglycol)COOH, hydroxybenzoates, hydroxyarylalkanoates and hydroxyalkylbenzoates. [148] Thus, in one example any divalent group that is 2 or more bonds in length, such as (CH₂)_n, where n is 2 or more, and polyethylene glycol may be used. The derivatized support can then be reacted with the desired peptide to capture the peptide on the support with loss of RιOH. The peptide may be reacted directly with the ester group in embodiments where COORi is an active ester group. In these examples, R-i is selected from groups, such as, but not limited to, Λ/-succinimidyl, sodium 3-sulfo-Λ/-succinimidyl and 4-nitrophenyl. [149] In other examples, the ester is saponified, e.g., with hydroxide, to provide the corresponding acid. This acid is then coupled with the amino-terminus of the peptide under standard peptide coupling conditions, e.g., 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The captured peptide is then truncated (fragmented) by reaction with an enzyme or reagent specific for a given amide bond of the peptide. Cleavage of the truncated peptide, containing an Λ/-terminal fragment of the original peptide, from the support is then accomplished by reaction with mild acid. Acids suitable for this cleavage include, but are not limited to, acetic acid, trifluoroacetic acid, para-toluenesulfonic acid and mineral acids. One such acid is 3-hydroxypicolinic acid, which is also a suitable matrix for the subsequent MALDI analysis. The peptide may also be captured through the carboxy terminus by employing an amino-derivatized support. [150] The starting amino-derivatized support may be prepared by reacting a hydroxylated support surface with diisopropylysilyl dichloride and an aminoalcohol. Aminoalcohols that may be used include, but are not limited to, aminoalkanols, hydroxy(polythyleneglycol)NH2, hydroxyanilines, hydroxyarylalkylamines and hydroxyalkylanilined. Capture of the peptide by the amino-derivatized support is achieved by dehydrative coupling of the peptide with the amino group. Such peptide coupling conditions. are well-known to those of skill in the art.

The captured peptide may then be truncated, cleaved from the support, and analyzed.

[151] Other linkers may be useful in capturing peptides on supports for MALDI analysis.

For example, trityl-containing linkers, functionalized with either ester or amino moieties, may be used to capture peptides at the amino or carboxy terminus, respectively. Other linkers known to those of skill in art, e.g., photocleavable linkers, are also available for use in capturing the peptides on the support surface.

[152] Photocleavable linkers. Photocleavable linkers may also be used in various embodiments of this invention. These linkers contain o-nitrobenzyl moieties and phosphate linkages, which allow for complete photolytic cleavage of the conjugates within minutes upon

UV irradiation.

[153] The UV wavelengths used are selected so that the irradiation will not damage the polypeptides and generally are about 350-380 nm, usually about 365 nm.

[154] Preparation of the Photocleavable Linkers. Photocleavable linkers can be prepared by the methods described below, by minor modification of the methods by choosing the appropriate starting materials or by any other methods known to those of skill in the art. For example by alkylation of 5-hydroxy-2-nitrobenzaldehyde with an hydroxyalkyl halide, e.g., 3- hydroxypropyl bromide, followed by protection of the resulting alcohol, e.g., as a silyl ether, provides a 5-(gamma; -silyloxyalkoxy)-2-nitrobenzaldehyde. Addition of an organometallic to the aldehyde affords a benzylic alcohol. Organometallics that can be used include trialkylaluminurns, such as trimethylaluminum; borohydrides, such as sodium borohydride; or metal cyanides, such as potassium cyanide.

[155] In the case of the metal cyanides, the product of the reaction, a cyanohydrin, is hydrolyzed under either acidic or basic conditions in the presence of either water or an alcohol to afford the compounds of interest.

[156] The silyl group of the side chain of the resulting benzylic alcohols can be exchanged for a 4,4'-dimethoxytriyl group by desilylation using, e.g., tetrabutylammonium fluoride, to give the corresponding alcohol, followed by reaction with 4,4'-dimethoxytrityl chloride.

[157] Chemically Cleavable Linkers. A variety of chemically cleavable linkers also can be used to link a polypeptide to a solid support. Acid-labile linkers are particularly useful chemically cleavable linkers for mass spectrometry, especially for MALDI-TOF, because the acid labile bond is cleaved during conditioning of the target polypeptide upon addition of a 3-

HPA matrix solution. The acid labile bond can be introduced as a separate linker group, e.g., an acid labile trityl group, or can be incorporated in a synthetic linker by introducing one or more silyl bridges using diisopropylysilyl, thereby forming a diisopropylysilyl linkage between the polypeptide and the solid support. The diisopropylysilyl linkage can be cleaved using mildly acidic conditions, such as 1.5% trifluoroacetic acid (TFA) or 3-HPA/1 % TFA MALDI-TOF matrix solution. Methods for the preparation of diisopropylysilyl linkages and analogs thereof are well-known in the art. See, e.g., Saha et al, J Org Chem, Vol. 58, pp. 7827-7831 (1993).

[158] A polypeptide of interest can be conjugated to a solid support, such as a bead. In addition, a first solid support such as a bead also can be conjugated, if desired, to a second solid support, which can be a second bead or other support, by any suitable means, including those disclosed herein for conjugation of a polypeptide to a support. Accordingly, any of the conjugation methods and means disclosed herein with reference to conjugation of a polypeptide to a solid support also can be applied for conjugation of a first support to a second support, where the first and second solid support can be the same or different. [159] Appropriate linkers, which can be cross-linking agents, for use for conjugating a polypeptide to a solid support include a variety of agents that can react with a functional group present on a surface of the support, or with the polypeptide, or both. Reagents useful as cross-linking agents include homo-bi-functional and, in particular, hetero-bi-functional reagents. Useful bi-functional cross-linking agents include, but are not limited to, A/-SIAB, dimaleimide, DTNB, Λ/-SATA, Λ/-SPDP, SMCC and 6-HYNIC. [160] A cross-linking agent can be selected to provide a selectively cleavable bond between a polypeptide and the solid support. For example, a photolabile cross-linker, such as 3-amino-(2-nitrophenyl)propionic acid (see Brown et al, Mol Divers, pp. 4-12 (1995); Rothschild et al, Nucl Acids Res, Vol. 24, pp. 351-66 (1996); and U.S. Patent No. 5,643,722) can be employed as a means for cleaving a polypeptide from a solid support. [161] Other cross-linking reagents are well-known in the art. See, e.g., Wong (1991), supra; and Hermanson (1996), supra.

[162] A polypeptide can be immobilized on a solid support, such as a bead, through a covalent amide bond formed between a carboxyl group functionalized bead and the amino terminus of the polypeptide or, conversely, through a covalent amide bond formed between an amino group functionalized bead and the carboxyl terminus of the polypepotide. [163] In addition, a bi-functional trityl linker can be attached to the support, e.g., to the 4-nitrophenyl active ester on a resin, such as a Wang resin, through an amino group or a carboxyl group on the resin via an amino resin. Using a bi-functional trityl approach, the solid support can require treatment with a volatile acid, such as formic acid or trifluoracetic acid to ensure that the polypeptide is cleaved and can be removed. In such a case, the polypeptide can be deposited as a beadless patch at the bottom of a well of a solid support or on the flat surface of a solid support. After addition of a matrix solution, the polypeptide can be desorbed into a MS.

[164] Hydrophobic trityl linkers also can be exploited as acid-labile linkers by using a volatile acid or an appropriate matrix solution, e.g., a matrix solution containing 3-HPA, to cleave an amino linked trityl group from the polypeptide. Acid lability also can be changed. For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be changed to the appropriate p-substituted, or more acid-labile tritylamine derivatives, of the polypeptide, i.e., trityl ether and tritylamine bonds to the can be made to the polypeptide. Accordingly, a polypeptide can be removed from a hydrophobic linker, e.g., by disrupting the hydrophobic attraction or by cleaving tritylether or tritylamine bonds under acidic conditions, including, if desired, under typical MS conditions, where a matrix, such as 3-HPA acts as an acid. [165] A polypeptide can be conjugated to a solid support, e.g., a bead, and the bead, either prior to, during or after conjugation of the polypeptide, can be conjugated to a second solid support, where one or both conjugations result in the formation of an acid-labile bond. For example, use of a trityl linker can provide a covalent or a hydrophobic conjugation, and, regardless of the nature of the conjugation, the trityl group is readily cleaved in acidic conditions.

[166] Orthogonally cleavable linkers also can be useful for binding a first solid support, e.g., a bead to a second solid support, or for binding a polypeptide of interest to a solid support. Using such linkers, a first solid support, e.g., a bead, can be selectively cleaved from a second solid support, without cleaving the polypeptide from the support; the polypeptide then can be cleaved from the bead at a later time. For example, a disulfide linker, which can be cleaved using a reducing agent, such as DTT, can be employed to bind a bead to a second solid support, and an acid cleavable bi-functional trityl group could be used to immobilize a polypeptide to the support. As desired, the linkage of the polypeptide to the solid support can be cleaved first, e.g., leaving the linkage between the first and second support intact. Trityl linkers can provide a covalent or hydrophobic conjugation and, regardless of the nature of the conjugation, the trityl group is readily cleaved in acidic conditions.

[167] A bead, e.g., can be bound to a second support through a linking group, which can be selected to have a length and a chemical nature such that high density binding of the beads to the solid support, or high density binding of the polypeptides to the beads, is promoted. Such a linking group can have, e.g., "tree-like" structure, thereby providing a multiplicity of functional groups per attachment site on a solid support. Examples of such linking groups include polylysine, polyglutamic acid, penta-erythrole and ./7s-hydroxy- aminomethane.

[168] A polypeptide can be conjugated to a solid support, or a first solid support also can be conjugated to a second solid support, through a non-covalent interaction. For example, a magnetic bead made of a ferromagnetic material, which is capable of being magnetized, can be attracted to a magnetic solid support, and can be released from the support by removal of the magnetic field. Alternatively, the solid support can be provided with an ionic or hydrophobic moiety, which can allow the interaction of an ionic or hydrophobic moiety, respectively, with a polypeptide, e.g., a polypeptide containing an attached trityl group or with a second solid support having hydrophobic character.

[169] A solid support also can be provided with a member of a specific binding pair and, therefore, can be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, a bead coated with avidin or with streptavidin can be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or derivative of biotin, such as imino-biotin. [170] It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, biotin, e.g., can be incorporated into either a polypeptide or a solid support and, conversely, avidin or other biotin binding moiety would be incorporated into the support or the polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, a nucleotide sequence and its complementary sequence, an antibody and the antigen to which it interacts specifically, and other such pairs knows to those skilled in the art.

[171] Immobilization of one or more polypeptides of interest, particularly target polypeptides, facilitates manipulation of the polypeptides. For example, immobilization of the polypeptides to a solid support facilitates isolation of the polypeptides from a reaction, or transfer of the polypeptides during the performance of a series of reactions. As such, immobilization of the polypeptides can facilitate conditioning the polypeptides or mass modification of the polypeptides prior to performing MS analysis.

[172] Conditioning a Polypeptide. Conditioning of a polypeptide prior to MS can increase the resolution of a mass spectrum of the polypeptide, thereby facilitating determining the identity of a target polypeptide. A polypeptide can be conditioned, e.g., by treating the polypeptide with a cation exchange material or an anion exchange material, which can reduce the charge heterogeneity of the polypeptide, thereby reducing or eliminating peak broadening. In addition, contacting a polypeptide with an alkylating agent, such as alkyliodide, iodoacetamide, iodoethanol, or 2,3-epoxy-1-propanol, e.g., can prevent the formation of disulfide bonds in the polypeptide, thereby increasing resolution of a mass spectrum of the polypeptide. Likewise, charged amino acid side chains can be converted to uncharged derivatives by contacting the polypeptides with trialkylsilyl chlorides, thus reducing charge heterogeneity and increasing resolution of the mass spectrum. [173] There are also means of improving resolution, particularly for shorter peptides, by incorporating modified amino acids that are more basic than the corresponding unmodified residues. Such modification in general increases the stability of the polypeptide during MS analysis. Also, cation exchange chromatography, as well as general washing and purification procedures which remove proteins and other reaction mixture components away from the target polypeptide, can be used to clean up the peptide after in vitro translation and thereby increase the resolution of the spectrum resulting from MS analysis of the target polypeptide.

[174] Conditioning also can involve incorporating modified amino acids into the polypeptide, e.g., mass modified amino acids, which can increase resolution of a mass spectrum. For example, the incorporation of a mass modified leucine residue in a polypeptide of interest can be useful for increasing the resolution, e.g., by increasing the mass difference, of a leucine residue from an isoleucine residue, thereby facilitating determination of an amino acid sequence of the polypeptide. A modified amino acid also can be an amino acid containing a particular blocking group, such as those groups used in chemical methods of amino acid synthesis. For example, the incorporation of a glutamic acid residue having a blocking group attached to the side chain carboxyl group can mass modify the glutamic acid residue and, provides the additional advantage of removing a charged group from the polypeptide, thereby further increasing resolution of a mass spectrum of a polypeptide containing the blocked amino acid.

[175] Use of a Pin Tool to Immobilize a Polypeptide. The immobilization of a polypeptide of interest to a solid support using a pin tool can be particularly advantageous. Pin tools include those disclosed herein or otherwise known in the art. See, e.g., U.S. Application Serial Nos. 08/786,988 and 08/787,639; and International PCT Application No. WO 98/20166. [176] A pin tool in an array, e.g., a 4 * 4 array, can be applied to wells containing polypeptides of interest. Where the pin tool has a functional group attached to each pin tip, or a solid support, e.g., functionalized beads or paramagnetic beads, are attached to each pin, the polypeptides in a well can be captured (1 pmol capacity).

[177] During the capture step, the pins can be kept in motion (vertical, 1-2 mm travel) to increase the efficiency of the capture. Where a reaction, such as an in vitro transcription is being performed in the wells, movement of the pins can increase efficiency of the reaction. [178] Polypeptides of interest, particularly target polypeptides, can be immobilized due to contact with the pin tool. Further immobilization can result by applying an electrical field to the pin tool. When a voltage is applied to the pin tool, the polypeptides are attracted to the anode or the cathode, depending on their net charge. Such a system also can be useful for isolating the polypeptides, since uncharged molecules remain in solution and molecules having a charge opposite to the net charge of the polypeptides are attracted to the opposite pole (anode or cathode). For more specificity, the pin tool (with or without voltage) can be modified to have conjugated thereto a reagent specific for the polypeptide of interest, such that only the polypeptides of interest are bound by the pins. For example, the pins can have nickel ions attached, such that only polypeptides containing a polyhistidine sequence are bound. Similarly, the pins can have antibodies specific for a target polypeptide attached thereto, or to beads that, in turn, are attached to the pins, such that only the target polypeptides, which contain the epitope recognized by the antibody, are bound by the pins. [179] Different pin conformations include, e.g., a solid pin configuration, or pins with a channel or with a hole through the centre, which can accommodate an optic fibre for mass spectrometer detection. The pin can have a flat tip or any of a number of configurations, including nanowell, concave, convex, truncated conic or truncated pyramidal, e.g., a size 4-800 micrometers across *100 micrometers in depth. The individual pins, which can be any size desired, generally are as long as about 10 millimeters, usually about 5 millimeters long, and particularly about 1 millimeters long.

[180] The pins and mounting plate can be made of polystyrene, which can be one piece injection moulded. Polystyrene is convenient for this use because it can be functionalized readily and can be moulded to very high tolerances.

[181] The pins in a pin tool apparatus can be collapsible, e.g., controlled by a scissor-like mechanism, so that the pins can be brought into closer proximity, reducing the overall size. [182] Captured polypeptides can be analyzed by a variety of means including, e.g., spectrometric techniques, such as UV/VIS, IR, fluorescence, chemiluminescence, NMR spectroscopy, MS or other methods known in the art, or combinations thereof. [183] If conditions preclude direct analysis of captured polypeptides, the polypeptides can be released or transferred from the pins, under conditions such that the advantages of sample concentration are not lost. Accordingly, the polypeptides can be removed from the pins using a minimal volume of eluent, and without any loss of sample. Where the polypeptides are bound to the beads attached to the pins, the beads containing the polypeptides can be removed from the pins and measurements made directly from the beads.

[184] Prior to determining the identity of a target polypeptide by MS, a pin tool having the polypeptide attached thereto can be withdrawn and washed several times, e.g., in ammonium citrate to condition the polypeptide prior to addition of matrix. The pins then can be dipped into matrix solution, with the concentration of matrix adjusted such that matrix solution adheres only to the very tips of the pins.

[185] Alternatively, the pin tool can be inverted and the matrix solution sprayed onto the tip of each pin using a microdrop device. The polypeptides also can be cleaved from the pins, e.g., into a nanowell on a chip, prior to addition of matrix. For analysis directly from the pins, a stainless steel "mask" probe can be fitted over the pins, then the mask probe can be installed in the MS.

[186] Two MS geometries can be used for accommodating a pin tool apparatus. A first geometry accommodates solid pins. In effect, the laser ablates a layer of material from the surface of the crystals, such that the resultant ions are accelerated and focused through the ion optics. A second geometry accommodates fibre optic pins, in which the laser strikes the samples from behind. In effect, the laser is focused onto the pin tool back plate and into a short optical fibre about 100 micrometers in diameter and about 7 millimeters in length to include thickness of the back plate. This geometry requires that the volatilized sample go through the depth of the matrix/bead mix, slowing and cooling down the ions and resulting in a type of delayed extraction, which can increase the resolution of the analysis. See, e.g., uhasz et al, Analysis, Anal Chem, Vol. 68, pp. 941-946 (1996), and see also, e.g., U.S. Patent Nos. 5,777,325; 5,742,049; 5,654,545; 5,641,959; 5,654,545 and 5,760,393 for descriptions of MALDI and delayed extraction protocols (incorporated by reference herein in their entirety). [187] The probe through which the pins are fitted also can be of various geometries. For example, a large probe with multiple holes, one for each pin, can be fitted over the pin tool and the entire assembly is translated in the X-Y axes in the MS. The probe also can be a fixed probe with a single hole, which is large enough to give an adequate electric field, but small enough to fit between the pins. The pin tool then is translated in all three axes, with each pin being introduced through the hole for sequential analyses. This latter format is more suitable for a higher density pin tool, e.g., a pin tool based on a 384 well or higher density microplate format. These two probes are suitable for the two MS geometries, as disclosed above.

[188] Pin tools can be useful for immobilizing polypeptides of interest in spatially addressable manner on an array. Such spatially addressable or pre-addressable arrays are useful in a variety of processes, including, for example, quality control and amino acid sequencing diagnostics. The pin tools described in the U.S. Application Nos. 08/786,988 and 08/787,639 and International PCT Application No. WO 98/20166 are serial and parallel dispensing tools that can be employed to generate multi-element arrays of polypeptides on a surface of the solid support. The array surface can be flat, with beads or geometrically altered to include wells, which can contain beads. A pin tool that allows the parallel development of a sample array is provided. Such a tool is an assembly of vesicle elements, or pins, where each of the pins can include a narrow interior chamber suitable for holding nanoliter volumes of fluid. Each of the pins fits inside a housing that has an interior chamber. The interior housing can be connected to a pressure source that can control the pressure within the interior housing chamber to regulate the flow of fluid through the interior chamber of the pins, thereby allowing for the controlled dispensing of defined volumes of fluid from the vesicles.

[189] The pin tool also can include a jet assembly, which can include a capillary pin having an interior chamber, and a transducer element mounted to the pin and capable of driving fluid through the interior chamber of the pin to eject fluid from the pin. In this way, the tool can dispense a spot of fluid to a support surface by spraying the fluid from the pin. The transducer also can cause a drop of fluid to extend from the capillary so that fluid can be passed to the array, or other solid support, by contacting the drop to the surface of the array. The pin tool also can form an array of polypeptides by dispensing the polypeptides in a series of steps, while moving the pin to different locations above the array surface to form the sample array. The pin tool then can pass prepared polypeptide arrays to a plate assembly that disposes the arrays for analysis by MS, which generates a set of spectra signal indicative of the composition of the polypeptides under analysis. [190] The pin tool can include a housing having a plurality of sides and a bottom portion having formed therein a plurality of apertures, the walls and bottom portion of the housing defining an interior volume; one or more fluid transmitting vesicles, or pins, mounted within the apertures, having a nanovolume sized fluid holding chamber for holding nanovolumes of fluid, the fluid holding chamber being disposed in fluid communication with the interior volume of the housing, and a dispensing element that is in communication with the interior volume of the housing for selectively dispensing nanovolumes of fluid form the nanovolume sized fluid transmitting vesicles when the fluid is loaded with the fluid holding chambers of the vesicles. This allows the dispensing element to dispense nanovolumes of the fluid onto the surface of the support when the apparatus is disposed over and in registration with the support.

[191] The fluid transmitting vesicle can have an open proximal end and a distal tip portion that extends beyond the housing bottom portion when mounted within the apertures. In this way the open proximal end can dispose the fluid holding chamber in fluid communication with the interior volume when mounted with the apertures. Optionally, the plurality of fluid transmitting vesicles are removably and replaceably mounted within the apertures of the housing, or alternatively can include a glue seal for fixedly mounting the vesicles within the housing.

[192] The fluid holding chamber also can include a narrow bore, which is dimensionally adapted for being filled with the fluid through capillary action, and can be sized to fill substantially completely with the fluid through capillary action. The plurality of fluid transmitting vesicles includes an array of fluid delivering needles, which can be formed of metal, glass, silica, polymeric material or any other suitable material, and, thus, as disclosed herein, also can serve as a solid support.

[193] The housing also can include a top portion, and mechanical biasing elements for mechanically biasing the plurality of fluid transmitting vesicles into sealing contact with the housing bottom portion. In addition, each fluid transmitting vesicle can have a proximal end portion that includes a flange, and further includes a seal element disposed between the flange and an inner surface of the housing bottom portion for forming a seal between the interior volume and an external environment. The biasing elements can be mechanical and can include a plurality of spring elements each of which are coupled at one end to the proximal end of each of the plurality of fluid transmitting vesicles, and at another end to an inner surface of the housing top portion. The springs can apply a mechanical biasing force to the vesicle proximal end to form the seal.

[194] The housing also can include a top portion, and a securing element for securing the housing top portion to the housing bottom portion. The securing element can include a plurality of fastener-receiving apertures formed within one of the top and bottom portions of the housing, and a plurality of fasteners for mounting within the apertures for securing together the housing top and bottom portions.

[195] The dispensing element can include a pressure source fluidly coupled to the interior volume of the housing for disposing the interior volume at a selected pressure condition. Moreover, where the fluid transmitting vesicles are to be filled through capillary action, the dispensing element can include a pressure controller that can vary the pressure source to dispose the interior volume of the housing at varying pressure conditions. This allows the controller varying element to dispose the interior volume at a selected pressure condition sufficient to offset the capillary action to fill the fluid holding chamber of each vesicle to a predetermined height corresponding to a predetermined fluid amount. [196] Additionally, the controller can include a fluid selection element for selectively discharging a selected nanovolume fluid amount from the chamber of each the vesicles. In addition, a pressure controller that operates under the controller of a computer program operating on a data processing system to provide variable control over the pressure applied to the interior chamber of the housing is provided.

[197] The fluid transmitting vesicle can have a proximal end that opens onto the interior volume of the housing, and the fluid holding chamber of the vesicles are sized to substantially completely fill with the fluid through capillary action without forming a meniscus at the proximal open end. Optionally, the apparatus can have plural vesicles, where a first portion of the plural vesicles include fluid holding chambers of a first size and a second portion including fluid holding chambers of a second size, whereby plural fluid volumes can be dispensed.

[198] The tool also can include a fluid selection element that has a pressure source coupled to the housing and in communication with the interior volume for disposing the interior volume at a selected pressure condition, and an adjustment element that couples to the pressure source for varying the pressure within the interior volume of the housing to apply a positive pressure in the fluid chamber of each the fluid transmitting vesicle to vary the amount of fluid dispensed therefrom. The selection element and adjustment element can be computer programs operating on a data processing system that directs the operation of a pressure controller connected to the interior chamber.

[199] The pin tool apparatus can be used for dispensing a fluid containing a polypeptide of interest, particularly a target polypeptide, into one or more wells of a multi-well device, which can be a solid support. The apparatus can include a housing having a plurality of sides and a bottom portion having formed therein a plurality of apertures, the walls and bottom portion defining an interior volume, a plurality of fluid transmitting vesicles, mounted within the apertures, having a fluid holding chamber disposed in communication with the interior volume of the housing, and a fluid selection and dispensing means in communication with the interior volume of the housing for variably selecting an amount of the fluid loaded within the fluid holding chambers of the vesicles to be dispensed from a single set of the plurality of fluid transmitting vesicles. Accordingly, the dispensing means dispenses a selected amount of the fluid into the wells of the multi-well device when the apparatus is disposed over and in registration with the device.

[200] The fluid dispensing apparatus for dispensing fluid containing a polypeptide of interest into one or more wells of a multi-well device can include a housing having a plurality of sides and top and bottom portions, the bottom portion having formed therein a plurality of apertures, the walls and top and bottom portions of the housing defining an interior volume, a plurality of fluid transmitting vesicles, mounted within the apertures, having a fluid holding chamber sized to hold nanovolumes of the fluid, the fluid holding chamber being disposed in fluid communication with the volume of the housing, and mechanical biasing element for mechanically biasing the plurality of fluid transmitting vesicles into sealing contact with the housing bottom portion.

[201] Determining the mass of the polypeptide by MS. The identity of an isolated target polypeptide is determined by MS. For MS analysis, the target polypeptide can be solubilised in an appropriate solution or reagent system. The selection of a solution or reagent system, e.g., an organic or inorganic solvent, will depend on the properties of the target polypeptide and the type of MS performed, and is based on methods well-known in the art. See, e.g., Vorm et al, Anal Chem, Vol. 61, p. 3281 (1994) for MALDI; and Valaskovic et al, Anal Chem, Vol. 67, p. 3802 (1995), for ESI. MS of peptides also is described, e.g., in International PCT Application No. WO 93/24834 to Chait et al. and U.S. Patent No. 5,792,664.

[202] A solvent is selected so as to considerably reduce or fully exclude the risk that the target polypeptide will be decomposed by the energy introduced for the vaporization process. A reduced risk of target polypeptide decomposition can be achieved, e.g., by embedding the sample in a matrix, which can be an organic compound such as a sugar, e.g., a pentose or hexose, or a polysaccharide such as cellulose. Such compounds are decomposed thermolytically into CO₂ and H₂O such that no residues are formed that can lead to chemical reactions. The matrix also can be an inorganic compound, such as nitrate of ammonium, which is decomposed essentially without leaving any residue. Use of these and other solvents is known to those of skill in the art. See, e.g., U.S. Patent No. 5,062,935. [203] MS formats for use in analyzing a target polypeptide include ionization (I) techniques, such as, but not limited to, MALDI, continuous or pulsed ESI and related methods, such as ionspray or thermospray, and massive cluster impact (MCI). Such ion sources can be matched with detection formats, including linear or non-linear reflectron TOF, single or multiple quadrupole, single or multiple magnetic sector, Fourier transform ion cyclotron resonance (FTICR), ion trap and combinations thereof such as ion-trap/TOF. For ionization, numerous matrix/wavelength combinations (MALDI) or solvent combinations (ESI) can be employed. Sub-attomole levels of protein have been detected, e.g., using ESI MS (see Valaskovic et al, Science, Vol. 273, pp. 1199-1202 (1996)) and MALDI MS (see Li et al, JAm Chem Soc, Vol. 118, pp. 1662-1663 (1996)).

[204] Electrospray MS has been described by Fenn et al, J Phys Chem, Vol. 88, pp. 4451-4459 (1984); and PCT Application No. WO 90/14148; and current applications are summarized in review articles. See Smith et al, Anal Chem, Vol. 62, pp. 882-89 (1990); and Ardrey, Spectroscopy, Vol. 4, pp. 10-18 (1992). MALDI-TOF: MS has been described by Hillenkamp et al, Burlingame and McCloskey, Eds., Elsevier Science Publ., pp. 49-60 (1990). With ESI, the determination of molecular weights in femtomole amounts of sample is very accurate due to the presence of multiple ion peaks, all of which can be used for mass calculation.

[205] The mass of a target polypeptide determined by MS can be compared to the mass of a corresponding known polypeptide. For example, where the target polypeptide is a mutant protein, the corresponding known polypeptide can be the corresponding normal protein.

[206] Similarly, where the target polypeptide is suspected of being translated from a gene having an abnormally high number of trinucleotide repeats, the corresponding known polypeptide can be the corresponding protein having a wild type number of repeats, if any. Where the target polypeptide contains a number of repeated amino acids directly correlated to the number of trinucleotide repeats transcribed and translated from DNA, the number of repeated trinucleotide repeats in the DNA encoding the polypeptide can be deduced from the mass of the polypeptide. if desired, a target polypeptide can be conditioned prior to MS, as disclosed herein, thus facilitating identification of the polypeptide.

[207] MALDI. Matrix Assisted Laser Desorption (MALDI) is one preferred method among the MS methods herein. Methods for performing MALDI are well-known to those of skill in the art. Numerous methods for improving resolution are also known. For example, resolution in MALDI-TOF-MS can be improved by reducing the number of high energy collisions during ion extraction. See, e.g., Juhasz et al. (1996) supra, see also, e.g., U.S. Pat. No. 5,777,325, 5,742,049, 5,654,545, 5,641 ,959, 5,654,545, 5,760,393 and 5,760,393 for descriptions of MALDI and delayed extraction protocols.

[208] Amino Acid Seguencing of Target Polypeptides. A process of determining the identity of a target polypeptide using mass spectrometry can be performed by determining the amino acid sequence, or a portion thereof, of a target polypeptide. Amino acid sequencing can be performed, e.g., from the carboxyl terminus using carboxypeptidase, such as carboxypeptidase Y, carboxypeptidase P, carboxypeptidase A, carboxypeptidase G or carboxypeptidase B, or other enzyme that progressively digests a polypeptide from its carboxyl terminus; or from the /V-terminus of the target polypeptide by using the Edman degradation method or using an aminopeptidase, such as alanine aminopeptidase, leucine aminopeptidase, pyroglutamate peptidase, dipeptidyl peptidase, microsomal peptidase or other enzyme that progressively digests a polypeptide from its amino terminus. [209] If desired, the target polypeptide first can be cleaved into peptide fragments using an enzyme, such as trypsin, chymotrypsin, Asp-N, thrombin or other suitable enzyme. The fragments then can be isolated and subjected to amino acid sequencing by mass spectrometry, or a nested set of deletion fragments of the polypeptide can be prepared by incubating the polypeptide for various periods of time in the presence of an aminopeptidase or a carboxypeptidase and, if desired, in the presence of reagents that modify the activity of a peptidase on the polypeptide. See, e.g., U.S. Patent No. 5,792,664; International Publication No. WO 96/36732.

[210] If desired, a tag, for example, a tag peptide, can be conjugated to a fragment of a target polypeptide. Such a conjugation can be performed prior to or following cleavage of the target polypeptide.

[211] Amino acid sequencing of a target polypeptide can be performed either on the free polypeptide or after immobilizing the polypeptide on a solid support. A target polypeptide can be immobilized on a solid support, e.g., by linking the polypeptide to the support through its amino terminus or its carboxyl terminus or directly or via a linker or [linkers by methods known to those of skill in the art or as described herein, then treating the immobilized polypeptide with an exopeptidase specific for the unbound terminus. For example, where a target polypeptide is linked to a solid support through its amino terminus, the immobilized polypeptide can be treated with a carboxypeptidase, which sequentially degrades the polypeptide from its carboxyl terminus. Alternatively, where the target polypeptide is linked to a solid support through its carboxyl terminus, the polypeptide can be digested from its amino terminus using, e.g., Edman's reagent.

[212] For amino acid sequencing, the target polypeptide is treated with the protease in a time-limited manner, and released amino acids are identified by MS. If desired, degradation of a target polypeptide can be performed in a reactor apparatus (see International Publication No. WO 94/21822, published September 29, 1994), in which the polypeptide can be free in solution and the protease can be immobilized, or in which the protease can be free in solution and the polypeptide can be immobilized.

[213] At time intervals or as a continuous stream, the reaction mixture containing a released amino acid is transported to a MS for analysis. Prior to MS analysis, the released amino acids can be transported to a reaction vessel for conditioning, which can be by mass modification. The determination of the amino acid sequence of the target polypeptide, particularly the identification of an allelic variation in the target polypeptide as compared to a corresponding known polypeptide, can be useful, e.g., to determine whether the subject from which the target polypeptide was obtained has or is predisposed to a particular disease or condition.

[214] If desired, the target polypeptide can be conditioned, e.g., by mass modified prior to sequencing. It should be recognized, however, that mass modification of a polypeptide prior to chemical or enzymatic degradation, for example, can influence the rate or extent of degradation. Accordingly, the skilled artisan will know that the influence of conditioning and mass modification on polypeptide degradation should be characterized prior to initiating amino acid sequencing. The process is conveniently performed in a multiplexing format, thereby allowing a determination of the identities of a plurality of two or more target polypeptides in a single procedure. For multiplexing, a population of target polypeptides can be synthesized by in vitro translation, where each of the target nucleic acids encoding each of the target polypeptides is translated, in a separate reaction, in the presence of one or more mass modifying amino acids. [215] The population of target polypeptides can be encoded, for example, by target nucleic acids representing the different polymorphic regions of a particular gene. Each of the individual reactions can be performed using one or more amino acids that are differentially mass modified, e.g., differentially mass modified, particularly using basic residues. Following translation, each target polypeptide is distinguishable by the particular mass modified amino acid.

[216] A plurality of target polypeptides also can be obtained, e.g., from naturally occurring proteins and examined by multiplexing, provided that each of the plurality of target polypeptides is differentially mass modified. For example, where a plurality of target polypeptides are being examined to determine whether a particular polypeptide is an allelic variant containing either a Gly residue or an Ala residue, the Gly and Ala residues in each polypeptide in the plurality can be mass modified with a mass label specific for that polypeptide.

[217] Identification of a Gly or Ala residue having a particular mass can be used to determine the particular polypeptide and the nature of the polymorphism.

[218] Amino acid modifications can be effected during or after in vitro translation of the target polypeptide. For example, any amino acid with a functional group on a side chain can be derivatized using methods known to those of skill in the art. For example, Λ/-succinimidyl-

3(2-pyridyldithio)propionate (SPDP) can be used to introduce sulfhydryl groups on lysine residues, thereby altering the mass of the polypeptide compared to the untreated polypeptide.

[219] Identifying the Polypeptide by Comparing the Mass of Target Polypeptide to a

Known Polypeptide. In methods other than those in which the polypeptide is sequenced and thereby identified, identification of the polypeptide is effected by comparison with a reference

(or known) polypeptide. The result indicative of identity is a function of the selected reference polypeptide. The reference polypeptide can be selected so that the target polypeptide will either have a mass substantially identical (identical within experimental error) to the reference polypeptide, or will have a mass that is different from the reference polypeptide.

[220] For example, if the reference polypeptide is encoded by a wild type allele of a gene that serves as a genetic marker, and the method is for screening for the presence of a disease or condition that is indicated by a mutation in that allele, then presence of the mutation will be identified by observing a difference between the mass of the target polypeptide and reference polypeptide. Observation of such difference thereby "identifies" the polypeptide and indicates the presence of the marker for the disease or condition. This result will indicate the presence of a mutation.

[221] Alternatively, if the reference polypeptide is encoded by a mutant allele of a gene that serves as a genetic marker, and the method is for screening for the presence of a disease or condition that is indicated by a mutation in that allele, then presence of the mutation will be identified by observing no difference between the mass of the target polypeptide and reference polypeptide. Observation of no difference thereby "identifies" the polypeptide and indicates the presence of the marker for the disease or condition. Furthermore, this result can provide information about the specific mutation. [222] Identifying a Target Polypeptide Based on Peptide Fragments of the Target Polypeptide. This process also provides a means for determining the identity of a target polypeptide by comparing the masses of defined peptide fragments of the target polypeptide with the masses of corresponding peptide fragments of a known polypeptide. Such a process can be performed, e.g., by obtaining the target polypeptide by in vitro translation, or by in vitro transcription followed by translation, of a nucleic acid encoding the target polypeptide; contacting the target polypeptide with at least one agent that cleaves at least one peptide bond in the target polypeptide, for example, an endopeptidase, such as trypsin or a chemical cleaving agent, such as cyanogen bromide, to produce peptide fragments of the target polypeptide; determining the molecular mass of at least one of the peptide fragments of the target polypeptide by MS; and comparing the molecular mass of the peptide fragments of the target polypeptide with the molecular mass of peptide fragments of a corresponding known polypeptide. The masses of the peptide fragments of a corresponding known polypeptide either can be determined in a parallel reaction with the target polypeptide, wherein the corresponding known polypeptide also is contacted with the agent; can be compared with known masses for peptide fragments of a corresponding known polypeptide contacted with the particular cleaving agent; or can be obtained from a database of polypeptide sequence information using algorithms that determine the molecular mass of peptide fragment of a polypeptide.

[223] This process of determining the identity of a target polypeptide by performing MS on defined peptide fragments of the target polypeptide is particularly adaptable to a multiplexing format. Accordingly, a process for determining the identity of each target polypeptide in a plurality of target polypeptides comprises, obtaining the plurality of target polypeptides; contacting each target polypeptide with at least one agent that cleaves at least one peptide bond in each target polypeptide to produce peptide fragments of each target polypeptide; determining the molecular mass of at least one of the peptide fragments of each target polypeptide in the plurality by MS; and comparing the molecular mass of the peptide fragments of each target polypeptide with the molecular mass of peptide fragments of a corresponding known polypeptide.

[224] In performing this process, it can be desirable to condition the target polypeptides. The polypeptides can be conditioned prior to cleavage, or the peptide fragments of the target polypeptide that will be examined by MS can be conditioned prior to MS. It also can be desirable to mass modify the target polypeptide, particularly to differentially mass modify each target polypeptide where a plurality of target polypeptides is being examined in a multiplexing format. Mass modification can be performed either on each polypeptide prior to contacting the polypeptide with the cleaving agent, or on the peptide fragments of the polypeptide that will examined by MS.

[225] A target polypeptide, particularly each target polypeptide in a plurality of target polypeptides, can be immobilized to a solid support prior to conditioning or mass modifying the polypeptide, or prior to contacting the polypeptide with a cleaving agent. In particular, the solid support can be a flat surface, or a surface with a structure, such as wells, such that each of the target polypeptides in the plurality can be positioned in an array, each at a particular address.

[226] In general, a target polypeptide is immobilized to the solid support through a cleavable linker, such as an acid labile linker, a chemically cleavable linker or a photocleavable linker. Following treatment of the target polypeptide, the released peptide fragments can be analyzed by mass spectrometry, or the released peptide fragments can be washed from the reaction and the remaining immobilized peptide fragment can be released, e.g., by chemical cleavage or photocleavage, as appropriate, and can be analyzed by MS. [227] It also can be useful to immobilize a particular target polypeptide to the support through both the amino terminus and the carboxyl terminus using, e.g., a chemically cleavable linker at one terminus and a photocleavable linker at the other end. In this way, the target polypeptides, which can be immobilized, e.g., in an array in wells, can be contacted with one or more agents that cleave at least one peptide bond in the polypeptides, the internal peptide fragments then can be washed from the wells, along with the agent and any reagents in the well, leaving one peptide fragment of the target polypeptide immobilized to the solid support through the chemically cleavable linker and a second peptide fragment, from the opposite end of the target polypeptide, immobilized through the photocleavable linker. Each peptide fragment then can be analyzed by mass spectrometry following sequential cleavage of the fragments, e.g., after first cleaving the chemically cleavable linker, then cleaving the photocleavable linker.

[228] Such a method provides a means of analyzing both termini of a polypeptide, thereby facilitating identification of the target polypeptide. Immobilization of a target polypeptide at both termini can be performed by modifying both ends of a target polypeptide, one terminus being modified to allow formation of a chemically cleavable linkage with the solid support and the other terminus being modified to allow formation of a photocleavable linkage with the solid support. Alternatively, the target polypeptides can be split into two portions, one portion being modified at one terminus to allow formation, for example, of a chemically cleavable linkage, and the second portion being modified at the other terminus to allow formation, e.g., of a photocleavable linkage. The two populations of modified target polypeptides then can be immobilized, together, on a solid support containing the appropriate functional groups for completing immobilization. Exemplary Uses

[229] Methods for determining the identity of a target polypeptide are disclosed herein. The identity of the target polypeptide allows information to be obtained regarding the DNA sequence encoding the target polypeptide. The target polypeptide can be from a eukaryote, such as a vertebrate, particularly a mammal such as a human, or can be from a prokaryote, including a bacterium or a virus. Generally, the target polypeptide can be from any organism, including a plant.

[230] A target polypeptide can be immobilized to a solid support, thereby facilitating manipulation of the polypeptide prior to MS. For example, a target polypeptide can be translated in vitro. Such a method of obtaining a target polypeptide conveniently allows attachment of a tag to the polypeptide, e.g., by producing a fusion polypeptide of the target polypeptide and a tag peptide, such as a polyhistidine tag. The presence of a tag peptide, such as a polyhistidine tag provides a means to isolate the target polypeptide, e.g., from the in vitro translation reaction, by passing the mixture over a nickel chelate column, since nickel ions interact specifically with a polyhistidine sequence. The target polypeptide then can be captured by conjugation to a solid support, thereby immobilizing the target polypeptide. If general, conjugation of the polypeptide to the solid support can be mediated through a linker, which provides desirable characteristics such as being readily cleavable, e.g., chemically cleavable, heat cleavable or photocleavable. For example, the target polypeptide can be immobilized at its amino terminus to a solid support through a diisopropylysilyl linker, which readily is cleavable under acidic conditions, such as when exposed to the MS matrix solution 3-HPA. In addition, the solid support, or a linker conjugated to the support or a group attached to such a linker, can be in the activated carboxy form, such as a sulfo-NHS ester, which facilitates conjugation of the polypeptide through its amino terminus. [231] Furthermore, conjugation of a polypeptide to a solid support can be facilitated by engineering the polypeptide to contain, e.g., a string of lysine residues, which increases the concentration of amino groups available to react with an activated carboxyl support. Of course, a polypeptide also can be conjugated through its carboxyl terminus using a modified linker, or can be conjugated using other linkers as disclosed herein or otherwise known in the art. The immobilized target polypeptide then can be manipulated, e.g., by proteolytic cleavage using an endopeptidase or a chemical reagent, such as cyanogen bromide, by sequential truncation from its free end using an exopeptidase or a chemical reagent, such as Edman's reagent, or by conditioning in preparation for MS analysis, e.g., by cation exchange to improve mass spectrometric analysis. An advantage of performing such manipulations with an immobilized polypeptide is that the reagents and undesirable reaction products can be washed from the remaining immobilized polypeptide, which then can be cleaved from the solid support in a separate reaction or can be subjected to MS, particularly MALDI-TOF, under conditions that cleave the polypeptide from the support, e.g., exposure of a polypeptide linked to the support through a photocleavable linker to the MALDI laser. [232] For purposes of the conjugation reactions, as well as enzymatic reactions, it is assumed that the termini of a target polypeptide are more reactive than the amino acid side groups due, e.g., to steric considerations. However, it is recognized that amino acid side groups can be more reactive than the relevant terminus, in which case the artisan would know that the side group should be blocked prior to performing the reaction of interest. Methods for blocking an amino acid side group are well-known and blocked amino acid residues are readily available and used, e.g., for chemical synthesis of peptides. Similarly, it is recognized that a terminus of interest of the polypeptide can be blocked due, e.g., to a post-translational modification, or can be buried within a polypeptide due to secondary or tertiary conformation.

[233] Accordingly, the artisan will recognize that a blocked amino terminus of a polypeptide, e.g., must be made reactive either by cleaving the amino terminal amino acid or by de-blocking the amino acid. In addition, where the terminus of interest is buried within the polypeptide structure, the artisan will know that the polypeptide, in solution, can be heated to about 70-100°C prior to performing a reaction. It is recognized, e.g., that when the reaction to be performed is an enzymatic cleavage, the enzymes selected should be stable at elevated temperatures. Such temperature stable enzymes, e.g., thermostable peptidases, including carboxypeptidases and aminopeptidases, are obtained from thermophilic organisms and are commercially-available. In addition, where it is desirable not to use heat to expose an otherwise buried terminus of a polypeptide, altering the salt conditions can provide a means to expose the terminus. For example, a polypeptide terminus can be exposed using conditions of high ionic strength, in which case an enzyme, such as an exopeptidase is selected based on its tolerance to high ionic strength conditions. [234] Measurement Methods . The experimental methods of this invention depend on measurements of gene expression products which have been determined to have utility as biomarkers. These markers may be mRNA or proteins in the body fluids or tissues of a patient. This section describes exemplary methods for measuring the cellular constituents in drug responses. This invention is adaptable to other methods of such measurement. [235] In one embodiment of this invention, the transcriptional state of the other cellular constituents is measured. The transcriptional state can be measured by techniques of hybridization to arrays of nucleic acid or nucleic acid mimic probes, described in the next subsection, or by other gene expression technologies, described in the subsequent subsection. However measured, the result is data including values representing mRNA abundance and/or ratios, which usually reflect DNA expression ratios (in the absence of differences in RNA degradation rates).

[236] In various alternative embodiments of the present invention, aspects of the biological state other than the transcriptional state, such as the translational state, the activity state or mixed aspects can be measured.

[237] In a particularly useful embodiment, the level of mRNA expression of the disclosed genes can be measured in a subject at various stages of the treatment with a EGFRI transcriptional or expression profile of the treatment over time. For example, mRNA transcripts corresponding to a gene disclosed herein.

[238] Transcriptional state measurement . Preferably, measurement of the transcriptional state is made by hybridization of nucleic acids to oligonucleotide arrays, which are described in this subsection. Certain other methods of transcriptional state measurement are described later in this subsection.

[239] Transcript arrays generally . In a preferred embodiment, the present invention makes use of "oligonucleotide arrays", also called herein "microarrays". Microarrays can be employed for analyzing the transcriptional state in a cell, and especially for measuring the transcriptional states of cells. [240] In one embodiment, transcript arrays are produced by hybridizing detectably- labelled polynucleotides representing the mRNA transcripts present in a cell, e.g., fluorescently-labelled cDNA synthesized from total cell mRNA or labelled cRNA, to a microarray. A microarray is a surface with an ordered array of binding, e.g., hybridization, sites for products of many of the genes in the genome of a cell or organism, preferably most or almost all of the genes. Microarrays can be made in a number of ways, of which several are described below. However produced, microarrays share certain characteristics. The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably the microarrays are small, usually smaller than 5 cm², and they are made from materials that are stable under binding, e.g., nucleic acid hybridization, conditions. A given binding site or unique set of binding sites in the microarray will specifically bind the product of a single gene in the cell. Although there may be more than one physical binding site, hereinafter "site", per specific mRNA, for the sake of clarity the discussion below will assume that there is a single site. In a specific embodiment, positionally addressable arrays containing affixed nucleic acids of known sequence at each location are used.

[241] It will be appreciated that when cDNA complementary to the RNA of a cell is made and hybridized to a microarray under suitable hybridization conditions, the level of hybridization to the site in the array corresponding to any particular gene will reflect the prevalence in the cell of mRNA transcribed from that gene. For example, when detectably- labelled, e.g., with a fluorophore, cDNA or cRNA complementary to the total cellular mRNA is hybridized to a microarray, the site on the array corresponding to a gene, i.e., capable of specifically binding the product of the gene, that is not transcribed in the cell will have little or no signal, e.g., fluorescent signal, and a gene for which the encoded mRNA is prevalent will have a relatively strong signal.

[242] Preparation of Microarrays. Microarrays are known in the art and consist of a surface to which probes that correspond in sequence to gene products, e.g., cDNAs, mRNAs, cRNAs, polypeptides and fragments thereof, can be specifically hybridized or bound at a known position. In one embodiment, the microarray is an array, i.e., a matrix, in which each position represents a discrete binding site for a product encoded by a gene, e.g., a protein or RNA, and in which binding sites are present for products of most or almost all of the genes in the organism's genome. In one embodiment, the "binding site", hereinafter "site", is a nucleic acid or nucleic acid analogue to which a particular cognate cDNA or cRNA can specifically hybridize. The nucleic acid or analogue of the binding site can be, e.g., a synthetic oligomer, a full-length cDNA, a less-than full-length cDNA, or a gene fragment. [243] Although in a preferred embodiment the microarray contains binding sites for products of all or almost all genes in the target organism's genome, such comprehensiveness is not necessarily required. The microarray may have binding sites for only a fraction of the genes in the target organism. However, in general, the microarray will have binding sites corresponding to at least about 50% of the genes in the genome, often at least about 75%, more often at least about 85%, even more often more than about 90% and most often at least about 99%. Preferably, the microarray has binding sites for genes relevant to testing and confirming a biological network model of interest. [244] As used herein, the term "gene" is identified as an open reading frame (ORF) of preferably at least 50, 75 or 99 nucleotides from which a mRNA is transcribed in the organism, e.g., if a single cell, or in some cell in a multicellular organism. The number of genes in a genome can be estimated from the number of mRNAs expressed by the organism, or by extrapolation from a well-characterized portion of the genome. When the genome of the organism of interest has been sequenced, the number of ORFs can be determined and mRNA coding regions identified by analysis of the DNA sequence. For example, the Saccharomyces cerevisiae genome has been completely sequenced and is reported to have approximately 6275 ORFs longer than 99 nucleotides. Analysis of these ORFs indicates that there are 5885 ORFs that are likely to specify protein products. See, e.g., Goffeau et al, Science, Vol. 274, pp. 546-567 (1996), which is incorporated by reference in its entirety for all purposes. In contrast, the human genome is estimated to contain approximately 25,000-35,000 genes.

[245] Preparing Nucleic Acids for Microarrays. As noted above, the "binding site" to which a particular cognate cDNA specifically hybridizes is usually a nucleic acid or nucleic acid analogue attached at that binding site. In one embodiment, the binding sites of the microarray are DNA polynucleotides corresponding to at least a portion of each gene in an organism's genome. These DNAs can be obtained by, e.g., PCR amplification of gene segments from genomic DNA, cDNA, e.g., by RT-PCR, or cloned sequences or the sequences may be synthesized de novo on the surface of the chip, e.g., by use of photolithography techniques, e.g., Affymetrix uses such a different technology to synthesize their oligos directly on the chip. PCR primers are chosen, based on the known sequence of the genes or cDNA, that result in amplification of unique fragments, i.e., fragments that do not share more than 10 bases of contiguous identical sequence with any other fragment on the microarray.

[246] Computer programs are useful in the design of primers with the required specificity and optimal amplification properties. See, e.g., Oligo pi version 5.0, Nat Biosci. In the case of binding sites corresponding to very long genes, it will sometimes be desirable to amplify segments near the 3' end of the gene so that when oligo-dT primed cDNA probes are hybridized to the microarray; less-than-full-length probes will bind efficiently. Typically each gene fragment on the microarray will be between about 20 bp and about 2000 bp, more typically between about 100 bp and about 1000 bp, and usually between about 300 bp and about 800 bp in length. PCR methods are well-known and are described, e.g., PCR Protocols: A Guide to Methods and Applications, Innis et al., Eds., Academic Press Inc., San Diego, CA (1990), which is incorporated by reference in its entirety for all purposes. It will be apparent that computer controlled robotic systems are useful for isolating and amplifying nucleic acids.

[247] An alternative means for generating the nucleic acid for the microarray is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using Λ/-phosphonate or phosphoramidite chemistries. See Froehler et al, Nucl Acid Res, Vol. 14, pp. 5399-5407 (1986); McBride et al, Tetrahedron Lett, Vol. 24, pp. 245-248 (1983). Synthetic sequences are between about 15 bases and about 500 bases in length, more typically between about 20 bases and about 50 bases. In some embodiments, synthetic nucleic acids include non- natural bases, e.g., inosine. As noted above, nucleic acid analogues may be used as binding sites for hybridization. An example of a suitable nucleic acid analogue is peptide nucleic acid. See, e.g., Egholm et al, Nature, Vol. 365, pp. 566-568 (1993); see also U.S. Patent No. 5,539,083.

[248] In an alternative embodiment, the binding (hybridization) sites are made from plasmid or phage clones of genes, cDNAs, e.g., expressed sequence tags, or inserts therefrom. See Nguyen et al, Genomics, Vol. 29, pp. 207-209 (1995). In yet another embodiment, the polynucleotide of the binding sites is RNA.

[249] Attaching Nucleic Acids to the Solid Surface. The nucleic acid or analogue are attached to a solid support, which may be made from glass, plastic, e.g., polypropylene and nylon, polyacrylamide, nitrocellulose or other materials. A preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al, Science, Vol. 270, pp. 467-470 (1995). This method is especially useful for preparing microarrays of cDNA. See, also, DeRisi er a/., Na. Genet, Vol. 14, pp. 457-460 (1996); Shalon et al, Genome Res, Vol. 6, pp. 639-645 (1996); and Schena et al., Proc Natl Acad Sci U S A, Vol. 93, pp. 10539-11286 (1995). Each of the aforementioned articles is incorporated by reference in its entirety for all purposes.

[250] A second preferred method for making microarrays is by making high-density oligonucleotide arrays. Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ (see Fodor et al, Science, Vol. 251, pp. 767-773 (1991 ); Pease et al, Proc Natl Acad Sci U S A, Vol. 91 , No. 11 , pp. 5022-5026 (1994); Lockhart et al, Nat Biotechnol, Vol. 14, p. 1675 (1996); and U.S. Patent Nos. 5,578,832; 5,556,752 and 5,510,270, each of which is incorporated by reference in its entirety for all purposes) or other methods for rapid synthesis and deposition of defined oligonucleotides. See Blanchard et al., Biosensors Bioelectron, Vol. 11 , pp. 687-690 (1996). When these methods are used, oligonucleotides, e.g., 25 mers, of known sequence are synthesized directly on a surface, such as a derivatized glass slide. Usually, the array produced is redundant, with several oligonucleotide molecules per RNA. Oligonucleotide probes can be chosen to detect alternatively spliced mRNAs. [251] Other methods for making microarrays, e.g., by masking (see Maskos and Southern, Nucl Acids Res, Vol. 20, pp. 1679-1684 (1992)), may also be used. In principal, any type of array, for example, dot blots on a nylon hybridization membrane (see Sambrook et al, Molecular Cloning-A Laboratory Manual, 2^nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), which is incorporated in its entirety for all purposes), could be used, although, as will be recognized by those of skill in the art, very small arrays will be preferred because hybridization volumes will be smaller. [252] Generating Labelled Probes. Methods for preparing total and poly(A)⁺ RNA are well-known and are described generally in Sambrook er al. (1989), supra. In one embodiment, RNA is extracted from cells of the various types of interest in this invention using guanidinium thiocyanate lysis followed by CsCI centrifugation. See Chirgwin er a/., Biochemistry, Vol. 18, pp. 5294-5299 (1979). Poly(A)⁺ RNA is selected by selection with oligo-dT cellulose. See Sambrook et al. (1989), supra. Cells of interest include wild-type cells, drug-exposed wild-type cells, cells with modified/perturbed cellular constituent(s), and drug-exposed cells with modified/perturbed cellular constituent(s).

[253] Labelled cDNA is prepared from mRNA or alternatively directly from RNA by oligo dT-primed or random-primed reverse transcription, both of which are well known in the art. See, e.g., Klug and Berger, Methods Enzymol, Vol. 152, pp. 316-325 (1987). Reverse transcription may be carried out in the presence of a dNTP conjugated to a detectable label, most preferably a fluorescently-labelled dNTP. Alternatively, isolated mRNA can be converted to labelled antisense RNA synthesized by in vitro transcription of double-stranded cDNA in the presence of labelled dNTPs. See Lockhart et al. (1996), supra, which is incorporated by reference in its entirety for all purposes.

[254] In alternative embodiments, the cDNA or RNA probe can be synthesized in the absence of detectable label and may be labelled subsequently, e.g., by incorporating biotinylated dNTPs or rNTP; or some similar means, e.g., photo-cross-linking a psoralen derivative of biotin to RNAs, followed by addition of labelled streptavidin, e.g., phycoerythrin- conjugated streptavidin; or the equivalent.

[255] When fluorescently-labelled probes are used, many suitable fluorophores are known, including fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others. See, e.g., Kricka, Nonisotopic DNA Probe Techniques, Academic Press, San Diego, CA (1992). It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.

[256] In another embodiment, a label other than a fluorescent label is used. For example, a radioactive label, or a pair of radioactive labels with distinct emission spectra, can be used. See Zhao et al, Gene, Vol. 156, p. 207 (1995); and Pietu et al., Genome Res., Vol. 6, p. 492 (1996). However, because of scattering of radioactive particles, and the consequent requirement for widely spaced binding sites, use of radioisotopes is a less-preferred embodiment.

[257] In one embodiment, labelled cDNA is synthesized by incubating a mixture containing 0.5 mM dGTP, dATP and dCTP plus 0.1 mM dTTP plus fluorescent deoxyribonucleotides (e.g., 0.1 mM Rhodamine 110 UTP (Perkin Elmer Cetus) or 0.1 mM Cy3 dUTP (Amersham)) with reverse transcriptase (e.g., ™ll, LTI Inc.) at 42°C for 60 minutes.

[258] Hybridization to Microarrays. Nucleic acid hybridization and wash conditions are chosen so that the probe "specifically binds" or "specifically hybridizes" to a specific array site, i.e., the probe hybridizes, duplexes or binds to a sequence array site with a complementary nucleic acid sequence but does not hybridize to a site with a non- complementary nucleic acid sequence. As used herein, one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch. Preferably, the polynucleotides are perfectly complementary (no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls. See, e.g., Shalon et al. (1996), supra; and Chee et al, supra.

[259] Optimal hybridization conditions will depend on the length, e.g., oligomer vs. polynucleotide greater than 200 bases; and type, e.g., RNA, DNA and PNA; of labelled probe and immobilized polynucleotide or oligonucleotide. General parameters for specific, i.e., stringent; hybridization conditions for nucleic acids are described in Sambrook et al. (1989), supra; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, NY (1987), which is incorporated in its entirety for all purposes. When the cDNA microarrays of Schena et al. (1995), supra, are used, typical hybridization conditions are hybridization in 5 x SSC plus 0.2% SDS at 65°C for 4 hours followed by washes at 25°C in low stringency wash buffer (1 x SSC plus 0.2% SDS) followed by 10 minutes at 25°C in high stringency wash buffer (0.1 x SSC plus 0.2% SDS). See Shena et al., Proc Natl Acad Sci U SA, Vol. 93, p. 10614 (1996). Useful hybridization conditions are also provided in, e.g., Hybridization With Nucleic Acid Probes, Tijessen, Ed., Elsevier Science Publishers B.V. (1993) and Kricka (1992), supra.

[260] Other Methods of Transcriptional State Measurement. The transcriptional state of a cell may be measured by other gene expression technologies known in the art. Several such technologies produce pools of restriction fragments of limited complexity for electrophoretic analysis, such as methods combining double restriction enzyme digestion with phasing primers (see, e.g., European Patent No. 0534858, filed September 24, 1992, by Zabeau et al), or methods selecting restriction fragments with sites closest to a defined mRNA end. See, e.g., Prashar and Weissman, Proc Natl Acad Sci U S A, Vol. 93, No. 2, pp. 659-663 (1996). Other methods statistically sample cDNA pools, such as by sequencing sufficient bases, e.g., 20-50 bases, in each of multiple cDNAs to identify each cDNA, or by sequencing short tags, e.g., 9-10 bases, which are generated at known positions relative to a defined mRNA end (see, e.g., Velculescu, Science, Vol. 270, pp. 484-487 (1995)), pathway pattern.

[261] Computer Implementations. In a preferred embodiment, the computation steps of the previous methods are implemented on a computer system or on one or more networked computer systems in order to provide a powerful and convenient facility for forming and testing models of biological systems. [262] The computer system may be a single hardware platform comprising internal components and being linked to external components. The internal components of this computer system include processor element interconnected with a main memory. For example, computer system can be an Intel Pentium based processor of 200 Mhz or greater clock rate and with 32 MB or more of main memory.

[263] The external components include mass data storage. This mass storage can be one or more hard disks (which are typically packaged together with the processor and memory). Typically, such hard disks provide for at least 1 GB of storage. Other external components include user interface device, which can be a monitor and keyboards, together with pointing device, which can be a "mouse", or other graphic input devices. [264] Typically, the computer system is also linked to other local computer systems, remote computer systems or wide area communication networks, such as the Internet. This network link allows the computer system to share data and processing tasks with other computer systems.

[265] Loaded into memory during operation of this system are several software components, which are both standard in the art and special to the instant invention. These software components collectively cause the computer system to function according to the methods of this invention. These software components are typically stored on mass storage. Alternatively, the software components may be stored on removable media such as floppy disks or CD-ROM (not illustrated). The software component represents the operating system, which is responsible for managing the computer system and its network interconnections. This operating system can be, e.g., of the Microsoft Windows family, such as Windows 95, Windows 98 or Windows NT, or a Unix operating system, such as Sun Solaris. Software includes common languages and functions conveniently present on this system to assist programs implementing the methods specific to this invention. Languages that can be used to program the analytic methods of this invention include C, C++, or, less preferably, JAVA.

[266] Most preferably, the methods of this invention are programmed in mathematical software packages, which allow symbolic entry of equations and high-level specification of processing, including algorithms to be used, and thereby freeing a user of the need to procedurally program individual equations or algorithms. Such packages include, e.g., MATLAB™ from Mathworks (Natick, MA), MATHEMATICA™ from Wolfram Research (Champaign, IL), and MATHCAD™ from Mathsoft (Cambridge, MA). [267] In preferred embodiments, the analytic software component actually comprises separate software components that interact with each other. Analytic software represents a database containing all data necessary for the operation of the system. Such data will generally include, but is not necessarily limited to, results of prior experiments, genome data, gene expression product data, experimental procedures and cost, and other information, which will be apparent to those skilled in the art.

[268] Analytic software includes a data reduction and computation component comprising one or more programs which execute the analytic methods of the invention. Analytic software also includes a user interface (Ul) which provides a user of the computer system with control and input of test network models, and, optionally, experimental data. The user interface may comprise a drag-and-drop interface for specifying hypotheses to the system. The user interface may also comprise means for loading experimental data from the mass storage component, e.g., the hard drive; from removable media, e.g., floppy disks or CD- ROM; or from a different computer system communicating with the instant system over a network, e.g., a local area network or a wide area communication network, such as the internet.

[269] This invention also provides a process for preparing a database comprising at least one of the markers set forth in this invention, e.g., mRNAs or protein products. For example, the polynucleotide or amino acid sequences are stored in a digital storage medium such that a data processing system for standardized representation of the genes that identify a breast cancer cell is compiled.

[270] The data processing system is useful to analyze gene expression by isolating polynucleotides or polypeptides from the cell, body fluid or tissue. The isolated polynucleotides are sequenced. The sequences from the sample are compared with the sequence(s) present in the database using homology search techniques. Greater than 90%, more preferably, greater than 95%, and more preferably, greater than, or equal to, 97%, sequence identity between the test sequence and the polynucleotides or polypeptides of the present invention, is a positive indication that the polynucleotide or polypeptide has been detected as defined above.

[271] Alternative computer systems and methods for implementing the analytic methods of this invention will be apparent to one of skill in the art. EXAMPLE

[272] This example demonstrates the discovery of biomarkers of drug response that could be used to screen patients to predict and evaluate the likelihood of developing or the development of liver and lung toxicity in patients treated with therapeutic agents. [273] The objective of this example was to purify and positively identify biomarkers of liver and lung toxicity that are relevant to treatment with therapeutic agents. Several candidate biomarkers were discovered that demonstrated the potential to distinguish patients that developed a grade 3 hepatotoxicity following treatment a therapeutic agent such as compound PKI166 from those that exhibited no adverse effect.

[274] PKI166 is (f?)-4-[(1-phenylethyl)amino]-7/-/-pyrrolo[2,3- ]pyrimidin-6-yl]-phenol and is a new chemical entity belonging to the pyrrolo-pyrimidine class of compounds. It is active in the low nanomolar range as an inhibitor of the EGFR tyrosine kinase and shows high selectivity against serine/threonine kinases and moderate selectivity against other tyrosine kinases. At the cellular level, PKI166 preferentially inhibits signalling through the ligand- activated EGFR signal transduction pathway. In addition, EGFR auto-phosphorylation is inhibited with an IC₅₀ value of approximately 10 nM. PKI166 is also active against the c- erbB2 tyrosine kinase with an IC₅₀ value between 0.10 and 1 μM. PKI166 exhibits potent anti-proliferative activity against the EGFR over-expressing A431 and BALB/MK and cell lines (IC₅₀ = 0.26 and 0.45 μM, respectively) and is efficacious in five different EGFR- dependent tumour models following oral administration (10-100 mg/kg/day). These include A-431 human epidermoid carcinoma (regressions at 100 mg/kg/day); NCI-H596 squamous cell lung carcinoma (early regressions at 100 mg/kg/day); DU-145 human prostate carcinoma; MDA MB-468 human breast carcinoma; and orthoptic 253J B-V bladder carcinoma (anti-tumour and anti-angiogenic activity). This example describes in detail the purification and identification of two of the candidate biomarkers; a protein at 11.7 kDa and a protein at 43 kDa: 1 ) The 11.7 kDa peak was identified as SAA protein. This protein was increased over the course of treatment in the group that developed hepatotoxicity. In addition, an 11.5 kDa protein, which co-purified with the 11.7 kDa protein, was identified as the "arginine-truncated form" of SAA protein. This form did not exhibit any change over the course of treatment. 2) The 43 kDa peak was identified as α-1 anti-chymotrypsin. This protein was expressed at lower levels before and during treatment in patients that developed grade 3 hepatotoxicity. [275] Plasma samples from patients that developed a grade 3 hepatotoxicity upon treatment with PKI166 were compared with plasma from matched controls that did not exhibit an adverse response to drug therapy. Plasma samples were collected at three time points during the study: pre-treatment, 15 days into the treatment and at the time of peak response. The exact time of peak response varied between patients, and each control was matched for the same time.

[276] In the discovery phase, plasma samples were fractionated on an anion exchange resin using a stepwise pH gradient and each fraction was then profiled under a variety of conditions. Univariate and pairwise analyses revealed candidate biomarkers that may predict which patients would develop hepatotoxicity before or during treatment as well as candidate biomarkers that correlated with the hepatotoxicity. The goal of this example was to identify two of the candidate biomarkers discovered in an earlier phase. The purification and identification of two candidate biomarkers is described.

[277] The identities of these proteins were determined by peptide mass fingerprinting using the PBS II ProteinChip Reader and confirmed by tandem MS analysis on a QSTAR

(ABI) equipped with PCI-1000 ProteinChip Interface.

Methods

[278] Samples. Several samples that had high and low levels of the 11.7 and 43.4 kDa proteins were selected for use in the purification and identification. These samples were combined for the purification of the 43.4 kDa protein. A larger quantity of starting material was required since this protein is present at relatively low abundance.

[279] Anion Exchange Fractionation. The plasma samples were first fractionated on an anion exchange resin. The number of fractions collected was nine in this purification/identification phase in order to ensure higher separation and purity of candidate markers. Since many proteins have the ability to associate with each other in serum, urea and detergent were added to the samples prior to fractionation to reduce protein-protein interactions.

[280] Samples were fractionated simultaneously in a 96 well micro titre filter plate according to the following protocol: 1. 100 μL of Q HyperD F resin (BioSepra) was added to each of eight wells and was equilibrated with 20 mM Tris-HCI, pH 9 buffer. 2. Samples were thawed on ice. Each sample was prepared by mixing 300 μL of sample and 86 μL of a buffer containing 9 M urea, 2% CHAPS, 100 mM Tris-HCI, pH 9. Total volume of each sample was 386 μL. For purification of the 43.4 kDa protein, 600 μL of the combined samples were used, buffers were scaled up proportionally but the amount of resin remained constant. 3. Samples were applied to the resin in wells of the titre plate, the plate was shaken gently for 5 minutes. The vacuum was turned on to allow liquid to flow to a recipient 96-well plate below. The flow-through fraction (fraction 1), containing unbound material, had a volume of -380 μL. 4. Each well was washed with an aliquot of 300 μL of 20 mM Tris-HCI, pH 9 buffer. The elution was the pH 9 fraction (fraction 2). The column-bound proteins were subsequently eluted by a stepwise pH gradient. 5. Each well was eluted with 300 μL of 20 mM Tris-HCI, pH 8 buffer. This was the pH 8 fraction (or fraction 3). 6. Each well was eluted with 300 μL of 20 mM Phosphate/Citrate, pH 7 buffer. This was the pH 7 fraction (or fraction 4). 7. Each well was eluted with 300 μL of 20 mM Phosphate/Citrate, pH 6 buffer. This was the pH 6 fraction (or fraction 5). 8. Each column was eluted with 300 μL of 20 mM Phosphate/Citrate, pH 5 buffer. This was the pH 5 fraction (or fraction 6). 9. Each column was eluted with 300 μL of 20 mM Phosphate/Citrate, pH 4 buffer. This was the pH 4 fraction (or fraction 7). 10. Each column was eluted with 300 μL of 20 mM Phosphate/Citrate, pH 3 buffer. This was the pH 3 fraction (or fraction 8). 11. Each column was eluted with 300 μL of 17% isopropanol / 33% ACN / 0.1% TFA solution. This was the "organic" fraction (or fraction 9). Proteins in each fraction, except fraction 1 , which contains high concentrations of urea and CHAPS, were detected through profiling on NP20 ProteinChip Arrays using 1.0 μL of each fraction. Proteins.in fraction 1 were detected by profiling on WCX arrays.

[281] Samples were stored at 4°C between purification steps. [282] Purification on Cation Exchange Resin. For the purification of the 43.4 kDa protein, the pH 3 fractions from the Q column were further fractionated on cation exchange columns. 1. 75 μL of S HyperD F resin (BioSepra) was added to each disposable plastic column (BioRad) and was equilibrated with 100 mM sodium acetate, pH 4, 0.1 M NaCI buffer. 2. The Q column-pH 3 fraction (100 μL) of each pooled patient sample was mixed with 300 μL of 100 mM sodium acetate, pH 4, 0.1 M NaCI buffer. 3. After 5 minutes, the diluted samples were applied to the column resin by gravity flow. The first flow-through was reapplied to the column to improve binding. The flow-through fraction (fraction S1), containing unbound material, had a volume of -400 μL. 4. Each column was washed with 200 μL of 100 mM sodium acetate, pH 4, 0.1 M NaCI. This elution was labelled fraction SF2. The column-bound proteins were subsequently eluted in a stepwise salt gradient. 5. Each column was eluted with 150 μL of 100 mM sodium acetate, pH 4, 0.2 M NaCI buffer. This was the SF3 fraction. 6. Each column was eluted with 150 μL of 100 mM sodium acetate, pH 4, 0.4 M NaCI buffer. This was the SF4 fraction. 7. Each column was eluted with 150 μL of 100 mM sodium acetate, pH 4, 0.6 M NaCI buffer. This was the SF5 fraction 8. Each column was eluted with 150 μL of 100 mM sodium acetate, pH 4, 0.8 M NaCI buffer. This was the SF6 fraction. 9. Finally, each column was eluted with 150 μL of 33% isopropanol / 17% acetonitrile solution. This was the "organic" fraction (or SF7).

[283] Size Fractionation. Proteins were separated based on their size using Microcon filtration units (Millipore). The YM30 Microcon units were used to separate the 11.7 kDa protein from larger proteins, especially human serum albumin. Since many smaller proteins bind to larger proteins or form oligomers in mild buffer conditions, they are retained with proteins larger than 30 kDa. Increasing amounts of acetonitrile (up to 80% ACN) and 0.1% TFA were used to disrupt protein-protein interactions. Proteins in the retentate and flow- through fractions were detected through profiling on NP20 ProteinChip Arrays using 1-2 μL of each fraction.

[284] For the purification of the 43.4 kDa protein, the SF3 fractions (-150 μL) of both samples were desalted and concentrated using YM10 Microcon filtration units. The concentrated samples (-15 μL) were washed twice with 100 μL of water to reduce the salt concentration prior to loading on a SDS-PAGE gel.

[285] Purification by SDS-PAGE Gel Electrophoresis. Once the proteins of interest were estimated to be sufficiently enriched and purified, the fractions were concentrated and loaded on SDS-PAGE gels for additional purification. For samples that had not been concentrated using Microcon filtration units, aliquots of 50-300 μL per fraction were concentrated in a Speed-Vac. Approximately 20 μL of concentrated material were loaded on the gel.

[286] Fractions containing the 11.7 kDa protein were run on both 16% Tricine gels (Invitrogen) and NuPAGE 4-12% Bis-Tris gels (Invitrogen). The fraction containing the 43.4 kDa protein was run on NuPAGE 4-12% Bis-Tris gels. Gels were stained with colloidal Coomassie dyes (Invitrogen). Protein bands corresponding to the biomarkers detected by SELDI-TOF-MS profiling were excised for further processing and identification. Gel pieces containing no protein were processed alongside the protein bands as a negative control. [287] Protein Identification. Protease Digestion and Peptide Mapping using the PBSII PROTEINCHIP™ Reader. The excised gel bands were treated to remove the Coomassie stain and SDS by incubating successively with methanol/acetic acid, ammonium bicarbonate (pH 8), and acetonitrile solutions. The gel pieces were dried in a Speedvac. The dried gel pieces were re-hydrated with 10 μL 25 mM ammonium bicarbonate (pH 8.0) containing 0.02 ug/μL modified Trypsin or AspN (Roche Applied Science). The digests were incubated for 3- 16 hours at 37°C and aliquots (1-2 μL) were removed at different time points for analysis. The supernatant containing the peptides from the protease digestion was applied to ProteinChip Arrays (H4 or NP20 arrays). A 1 μL aliquot of saturated CHCA in 50% acetonitrile, 0.5% trifluoroacetic acid was applied to each spot. [288] Data was collected in the peptide mass range (<10 kDa) on the PBSII PROTEINCHIP™ Reader. Spectra were internally calibrated with known peaks from protease autolysis or added calibrants. Only peptide masses of the unique peptides, after subtracting the background from the negative control (protease-processed gel pieces without proteins), were used for the subsequent database search. Molecular weights of peptides unique to each candidate biomarker were submitted to the NCBI and/or Swiss-Prot protein databases using the ProFound search algorithm (http://129.85.19.192/prowl- cgi/ProFound.exe) as the database mining tool. Protein Identification by Peptide Fragmentation using a Q-STAR Tandem MS Eguipped with a PCI-1000 ProteinChip Interface. The identification was further confirmed by subjecting specific peptides from the protease digest to sequence analysis by tandem MS. The single- MS spectra and CID spectra from MS/MS analysis were acquired on a Q-STAR™ mass spectrometer (ABI) equipped with a Ciphergen PCI-1000 ProteinChip Interface. An aliquot (1-2 μL) of the protease digest was spotted on a NP20 ProteinChip Array and 1 μL of saturated CHCA in 50% acetonitrile and 0.5% trifluoroacetic acid was applied to each spot. After the spots were dry, the ProteinChip Array was inserted into the ProteinChip Interface. Spectra were collected from 0-3 kDa in single-MS mode. Selected ions were introduced into the collision cell for CID fragmentation. The CID spectral data was submitted to the database-mining tool, UCSF Protein Prospector MS-Tag program (http://prospector.ucsf.edU/ucsfhtml3.4/mstagfd.htm), to identify the protein. Results

[289] Part A. Purification and Identification of the 11.7 kDa Protein. The 11.7 kDa protein exhibited a statistically significant increase over time in the patients that developed grade 3 hepatotoxicity. Several different protocols for the purification of the 11.7 kDa protein from plasma were tried. The most successful method is summarized below; [290] Protein purification and identification methods for the 11.7 kDa protein. Purification & ID scheme for the -11.7 kDa proteins Serum samples (Patients #121 and #216 Anion exchanger column fractionation (pH 9 to pH 3 and Organic buffers for elution) Organic fraction (or pH 5 fraction _SDS-PAGE) Membrane concentrator YM-30 (Wash the retentate with 40% to 80% acetonitrile + 0.1% TFA) Filtrate fraction (<30kDa in 80% ACN) 1-D SDS-PAGE gel (16% acrylamide, Tricine gel) Purified protein bands In-gel digestion with trypsin or AspN Protein IDby Peptide Mass Fingerprinting using PBS II and by Peptide CID using MS/MS with PCI-1000 [291] After Q column fractionation of eight samples, two samples, No. 121 CA, which contained a high level of the 11.7 kDa protein and No. 216 TA, which had a low level of this protein, were used to purify the 11.7 kDa protein. This protein was enriched in both the pH 5 and organic fractions from the Q column chromatography step. Several other proteins were observed in the 8-15 kDa mass range after anion exchange fractionation. [292] The pH 5 fraction also contained high concentrations of albumin (66 kDa). To separate the 11.7 kDa protein from other unwanted proteins, the pH 5 fractions were concentrated and further separated on a SDS / 4-12% acrylamide gel. The gel band corresponding to the 11.7 kDa protein was identified by comparing the gel profiles of samples with high and low levels of this protein.

[293] Like the 11.7 kDa peak, the 11.9 kDa peak exhibited an increase over time in patients which developed grade-3 hepatotoxicity. Further inspection of the 11.9 kDa peak suggests this peak is likely to be an SPA adduct of the 11.7 kDa peak since its mass corresponds to that expected for the 11.7 kDa peak plus one molecule of SPA (207 kDa). [294] In addition, it was found that the 11.9 kDa peak did not appear when CHCA was used as the matrix. Therefore, the gel band of interest contained both the 11.5 kDa and 11.7 kDa proteins. Since this relatively low percentage acrylamide gel does not resolve the 11.5 kDa and 11.7 kDa protein, the entire gel band around 11.7 kDa was isolated for in-gel trypsin digestion and protein identification.

[295] Identification of the purified 11.7 kDa Protein . After excising the band containing the 11.5 kDa and 11.7 kDa proteins, the gel pieces were treated (as described above) and digested overnight with modified trypsin. The peptide products were detected on a PBSII ProteinChip Reader. Higher accuracy internal mass calibration was performed and peaks associated with tryptic autodigestion were excluded from the subsequent database search. Only peaks which were unique to the proteins of interest were selected and submitted for protein identification by peptide mass fingerprinting using the ProFound database search engine (ProteoMetrics). Human serum amyloid A was the top candidate with a Z-score of 2.23, indicating a high confidence match.

TABLE 1 Protein Candidates Identified by the Peptide-Mass Fingerprinting Method (MS Using PBS II) and Confirmed by Peptide Fragmentation (MS/MS Using QSTAR) Marker Highest Protein MW ID bv PBS II ID bv MS/MS Protein Candidate(s) (kDa) Estimated Z-Score Seguenced Peptides # 1 Serum Amyloid A 11.68 2.23 SFFSFLGEAFDGAR (11.7 kDa) Protein (SAA) (7 peptides (SEQ ID NO: 5). matched) (1550.690 Da - Avg M) RGPGGAWAAEVISDAR (SEQ ID NO: 6). (1612.766 Da - Avg M) HFRPAGLPEKY (SEQ ID NO: 7). (MH+ = 1314.6959 Da - iso) # 2 Alpha-1 45.27 2.36 HPNSPLDEENLTQENQDR (43.4 kDa) Antichymotrypsin (8 peptides (SEQ ID NO: 8). matched) (2134.95 Da - Avg M) AVLDVFEEGTEASAATAVK (SEQ ID NO: 9). (1906.95 Da - Avg M)

Note: Z score below 1.28 corresponds to below 90 percentile. See below.

[296] Description ofZ Score (from ProFound site). ProFound calculates the probability that a candidate in a database search is the protein being analyzed. However, it is not easy to cast the calculated probability into the common language of traditional statistics. Here, as an indicator of the quality of the search result, a Z score is estimated when the search result is compared against an estimated random match population. Z score is the distance to the population mean in unit of standard deviation. It also corresponds to the percentile of the search in the random match population. For instance, a Z score of 1.65 for a search means that the search is in the 95^th percentile. In other words, there are about 5% of random matches that could yield higher Z scores than this search. Conceptually, this "95^th percentile" is different from "95% confidence" that the search is a correct identification. [297] The following is a list for Z score and their corresponding percentile in an estimated random match population: Z Percentile 1.282 90.0 1.645 95.0 2.326 99.0 3.090 99.9

[298] The identification process was extended by analyzing the trypsin digest on an Q- STAR™ tandem MS (ABI) equipped with a PCI-1000 Interface. The very same tryptic digest was analyzed and peptides with masses of 1314 kDa, 1612 kDa and 1550 kDa were selected to undergo collision-induced dissociation. The resulting ionic fragments were then submitted for protein identification using the prospector database search engine (from UCSF). In each method, the top candidate for the purified 11.5 kDa and 11.7 kDa proteins digested with trypsin was returned as the SAA protein. Based on the Protein Chip Reader profiling data, both 11.5 kDa and 11.7 kPa peaks have similar peak intensities in the anion exchange pH 5 fraction. As a result, both proteins are likely to be in the gel band in similar amounts. Since most of the detected peptides with a strong signal (7 out of 8) correlate with the top candidate, SAA, and since few of the remaining strong signals correlate with the next protein candidate, it was determined that the 11.5 kDa and 11.7 kDa proteins were different forms of the same protein.

[299] The trypsin-digested peptides of human SAA protein precursor that matched with the PBS ll-detected peptides are:

Mr(calc) Start-End Seguence 1456.578 66-80 GPGGVWAAEAISDAR (SEQ IP NO: 10) 1550.690 20-33 SFFSFLGRAFDGAR (SEQ IP NO: 11). 1612.766 65-80 RGPGGVWAAEAISDAR (SEQ IP NO: 12). 1640.819 109-122 DPNHFRPAGLPEKY (SEQ IP NO: 13). 1670.802 44-57 EANYIGSDKYFHAR (SEQ IP NO: 14). 1913.123 106-122 SGKDPNHFRPAGLPEKY (SEQ IP NO: 15). 2178.262 86-105 FFGHGAEDSLADQAANEWGR (SEQ IP NO: 16).

[300] The amino acid sequences of the 1550.690 Da and 1612.766 Da peptides were confirmed by MS/MS data. The 1314 Da peptide was also subjected to MS/MS sequence analysis and matched to SAA. This peptide did not have an Λ/-terminus matching that predicted from trypsin digestion, explaining why it did not match in the original ProFound database search.

[301] Determination of the Λ/-ter minal seguence of the 11.5 kDa and 11.7 kDa peaks.

Careful analysis of the sequence of SAA raised the possibility that the 11.5 kPa and

11.7 kPa peaks may be different forms of SAA, differing by a single arginine residue at the

Λ/-terminus.

[302] Predicted masses of the two forms of SAA are 11 ,526 Pa and 11 ,683 Pa, closely matching the observed masses. [303] Molecular Weights and Amino Acid Seguences of Pifferent Forms of Human Serum Amyloid A protein; Serum amyloid A protein precursor (SAA), 122 aa, 13.59 kPa. Mature protein - the signal in positions 1-18 has been removed: (P02735) Amyloid protein A (Amyloid fibril protein AA)

A. SERUM AMYLOIP A PROTEIN at positions 19 - 122 : MW (average mass): 11682.70, MW (monoisotopic mass): 11675.49 1 10 20 30 40 50 RSFFSFLGEA FPGARPMWRA YSPMREANYI GSPKYFHARG NYPAAKRGPG GVWAAEAISP ARENIQRFFG HGAEPSLAPQ AANEWGRSGK PPNHFRPAGL PEKY (SEQ IP NO: 17).

B. Arginine-truncated form of SERUM AMYLOIP A PROTEIN: MW (average mass): 11526.51 , MW (monoisotopic mass): 11519.39 (20-122): 1 10 20 30 40 50 SFFSFLGEAF PGARPMWRAY SPMREANYIG SPKYFHARGN YPAAKRGPGG VWAAEAISPA RENIQRFFGH GAEPSLAPQA ANEWGRSGKP PNHFRPAGLP EKY (SEQ IP NO: 18).

[304] To address this possibility, another digest was performed using AspN. Trypsin cleaves after arginine residues and a trypsin digestion of the 1 1.5 kPa and 11.7 kPa forms of SAA would yield identical profiles except for one free arginine residue, which is too small to be detected using matrix assisted MS methods. Therefore, AspN was chosen to cleave the proteins into fragments that would preserve the Λ/-terminus of the two proteins. A new purification of the 11.5 kPa and 11.7 kPa protein was performed using the organic fraction from the anion exchange fractionation which also contained these two proteins. This fraction was passed through a Microcon YM30 microfiltration membrane to improve the yield for the two proteins of interest. However, the 11.5 kPa and 11.7 kPa proteins did not filter through the 30,000 MW cut-off membrane when the sample was treated with relatively low concentrations of acetonitrile (less than 40% _V), suggesting these proteins may bind to other proteins or form oligomers. The two proteins passed through the filtration membrane after treatment with 80% acetonitrile, 0.1% TFA. [305] The YM30 filtrate contained very few proteins larger than 12 kPa. Although the

11.5 kPa andl 1.7 kPa proteins were the predominant species in this fraction, a significant number of smaller proteins (<10 kPa) were also present.

[306] Consequently, the YM30 filtrate was loaded on a high-resolution SPS gel, 16% acrylamide / tricine to achieve separation of the 11.5 kPa and 11.7 kPa proteins from other unwanted proteins and to resolve the 11.5 kPa and 11.7 kPa proteins from each other. After

Coomassie staining, two bands around 11.7 kPa were visible. The upper band corresponded to the 11.7 kDa protein, the lower band to the 11.5 kDa protein. Each band was carefully isolated from the gel and separately digested with AspN. Peptides resulting from each AspN digest were detected on the PBSII ProteinChip Reader as previously described.

[307] The peptide maps of the independent digests of the purified 11.5 kPa and 11.7 kPa proteins are very similar, supporting the theory that the 11.5 kPa and 11.7 kPa peaks are derived from the same protein. A database search using Profound yielded the same identification, SAA, in both cases. Sequence analysis of several peptides by tandem MS confirmed this identification

[308] SAA is synthesized as a 59 kDa precursor. The mature form of SAA (minus the leading sequence) has the predicted mass of 11683 Da, which is the 11.7 kDa protein. The

11.5 kDa peak is expected to be the "arginine-truncated" form of SAA with a predicted mass of 11526 Da.

[309] The AspN-digested peptides of human serum amyloid A protein precursor that matched with the PBS ll-detected peptides are:

Mr.calc) Start-End Seguence 1270.370 51-60 DKYFHARGNY (SEQ ID NO: 19). 1318.368 97-108 DQAANEWGRSGK (SEQ ID NO: 20). 1640.819 109-122 DPNHFRPAGLPEKY (SEQ ID NO: 21 ). 1655.831 61-77 DAAKRGPGGVWAAEAIS (SEQ ID NO: 22) 1746.860 78-92 DARENIQRFFGHGAE (SEQ ID NO: 23) 2133.265 78-96 DARENIQRFFGHGAEDSLA (SEQ ID NO: 24) 2941.172 97-122 DQAANEWGRSGKDPNHFRPAGLPEKY (SEQ ID NO: 25)

[310] The amino acid sequences of the 1640.8 Da and 1746.9 Da peptides were confirmed by MS/MS data.

[311] Inspection of the MS data collected on both a PBS II and a Q-STAR MS (ABI) revealed a 1307.4 Da peptide present mainly in the AspN digest of the 11.7 kDa protein, and a weak 1151.6 Da peptide primarily in the AspN digest of the 11.5 kDa protein. The mass of the 1307.4 Da peptide in the 11.7 kDa protein AspN digest is consistent with the Λ/-terminal

RSFFSFLGEAF fragment of SAA including an arginine residue at the Λ/-terminus. Similarly, the 1151.6 Da peptide in the 11.5 kDa protein is consistent with the Λ/-temninal

SFFSFLGEAF fragment of SAA lacking an Λ/-terminal arginine residue. The sequence of the 1307.4 Da peptide was confirmed by MS/MS.

[312] Purification and Identification of the 43.4 kDa Protein. Protein profiling revealed that the 43.4 kDa protein was expressed at lower levels in the patients which developed grade 3 hepatotoxicity than in the patients that had no adverse effect. The level of this protein remained constant prior to and during treatment.

[313] Purification of the 43.4 kDa Protein. Several different methods to purify this 43.4 kDa biomarker protein from plasma were tested and the most successful one is summarized below;

[314] Protein Purification and Identification Methods for the 43.4 kDa Protein. Purification

& IP scheme for the 43.4 kPa proteins Serum samples (Patients #112 [High] and #125[Low]) Anion exchanger column fractionation (Q-HyperP) (pH 9 to pH 3 and Organic buffers for elution) "pH 3" fraction Cation exchanger column fractionation (S-HyperP) (pH 4 buffer and NaCI step-gradient from 0.1 M to 0.8 M) "pH 4 + 0.2M NaCI" fraction 1-P SPS/PAGE gel (4-12% acrylamide, MES gel- NuPAGE) Purified 43.4 kPa protein band In-gel digestion with trypsin or AspN Protein IP by Peptide Mass Fingerprinting using PBS II and by Peptide CIP using MS/MS with PCI-1000 Pesalting/concentrating using YM-10 microcon

[315] Based on the results of protein profiling during the biomarker discovery phase, the Sample Nos. 42 and 43, which had high levels of the 43.4 kPa protein, and the Samples Nos. 16 and 17, which had low levels of the 43.4 kDa protein, were selected as starting materials for the purification of the 43.4 kDa protein. [316] This protein was enriched in the pH 3 fraction from the anion exchange Q-column chromatography but many unwanted proteins in the 35-55 kDa range were also present in this fraction. Additionally, this pH 3 fraction (Q-pH3) also contained high amounts of albumin (66 kDa).

[317] High concentrations of albumin in the fraction with lower abundance of the protein of interest can cause smearing on the gel. It is therefore important to reduce the amount of albumin prior to gel electrophoresis. To increase the purity of the 43.4 kDa protein, the Q- pH3 fraction was loaded on an S-Hyper D cation exchanger column (S-column) equilibrated in pH 4 buffer. Elution of the proteins was driven by a salt gradient. Ultimately, the 43.4 kDa protein eluted from the S column by applying a pH 4 buffer with 0.2 M NaCI. Analysis of this pH 4, 0.2 M NaCI fraction (SF3) with a ProteinChip Reader established that very little albumin, a major component of the Q-pH 3 fraction, was left in this fraction (see Figure 12). The SF3 fraction was de-salted, concentrated and loaded to a SDS, 4-12% acrylamide gel to achieve further separation.

[318] There were two major protein bands in the 38-50 kDa range as visualized by Coomassie staining of the gel. Comparison of the SF3 fraction MS profiles to the bands in the gel correlated the upper band to the 43.4 kDa protein, and the lower band to a protein that produced a broad MS peak covering from 34-39 kDa range (broad peaks are characteristic of glycosylated proteins). Both bands were carefully excised from the gel for digestion with trypsin.

[319] Identification of the Purified 43.4 kDa Protein. The gel-purified 43.4 kDa protein was digested with modified trypsin and the resulting peptide mixture was first analyzed on the PBSII ProteinChip Reader. Several peptides unique to the 43.4 kDa band were subsequently subjected to sequence analysis by tandem MS. In the detailed results of the tandem MS analysis, the top candidate with the highest confidence score for the identification of the 43.4 kDa protein, using data from both peptide mapping and tandem MS sequence analysis, is human α-1 AACT.

[320] The full-length AACT protein (processed form lacking its signal sequence) contains 397 amino acids and has a predicted mass of 45.27 kDa. The sequence is shown below. [321] Molecular Weights and Amino Acid Seguence of Human Alpha-1 Antichymotrypsin. The precursor form of human alpha-1 antichymotrypsin precursor (P01011 ) has a predicted molecular weight of 47.85 kDa. Once the leader sequence has been removed, the human alpha-1 antichymotrypsin is predicted to contain 397 amino acids, with a molecular weight of 45.27 kDa. The sequence of the mature form is shown below. Peptides that were detected after trypsin digestion are; 1-40, 122-154, 192-207, 245-260, 284-293, and 328-356. No peptides were detected at the C-terminus (peptides 357-397), suggesting a potential truncation of the C-terminal end.

1 * 10 * 20 * 30 * 40 * 50 l ::? s- ::?: ^τc^ Qr'FΛτι:v: .G:Λ Λ '^")"A";f ^QLVLKAPDKNV 51 IFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTETSEAEIHQSFQHLL 101 RTLNQSSDELQLSMGNAMFVKA-37 :. ^~T_T ft^'CI*. YG.^Λ7ΑT"Q;S 151 ASilKKLINDY/KNGTRGKITDLIKDLDSQTMMVLVNYIFFKli^EIIFi F 201 QDTHQSRFYLSKKKVMVPMMSLHHLTIPYFRDEELSCTVVELKXTS. S 251 ALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREΪGSLILFRFSISRDY 301 LNDILLQLGIEEAFTSKADLSGITGAR^'KL.H\^?SQn. HXAVIJDVFEEGTEAS 351 AkTAVKITLLSALVETRTIVRFNRPFLMIIVPTDTQNIFFMSKVT P (SEQ ID NO: 26)

[322] The predicted mass differs from the observed mass of 43.4 kDa, suggesting that AACT may be truncated. The detected tryptic peptides for α-1 AACT are:

Mr(calc) Start-End Seguence 1061.243 284-292 EIGELYLPK (SEQ ID NO: 27) 1094.279 328-337 NLAVSQVVHK (SEQ ID NO: 28) 1108.222 276-283 WRDSLEFR (SEQ ID NO: 29) 1664.833 122-135 EQLSLLDRFTEDAK (SEQ ID NO: 30) 1752.941 245-260 YTGNASALFTLPDQDK (SEQ ID NO: 31 ) 1773.903 194-207 WEMPFDPQDTHQSR (SEQ ID NO: 32) 1892.009 137-154 LYGSEAFATDFQDSAAAK (SEQ ID NO: 33) 1908.093 338-356 AVLDVFEEGTEASAATAVK (SEQ ID NO: 34) 1973.150 192-207 AKWEMPFDPQDTHQSR (SEQ ID NO: 35) 2136.176 1-18 HPNSPLDEENLTQENQDR (SEQ ID NO: 36) 2225.486 19-39 GTHVDLGLASANVDFAFSLYK (SEQ ID NO: 37) [323] These peptides cover several regions of the AACT protein, including the predicted N-terminus. No peptides were detected from the C-terminal region suggesting that the 43.4 kDa protein marker may be processed at the C-terminus. The 2136.897 Da and 1907.960 Pa peptides were chosen for fragmentation by tandem MS. The resulting fragment ions generated from each peptide in the collision cell were detected and their masses submitted for database search to identify the best candidate proteins for the 43.4 kDa protein. The top candidate for each of these peptides was human α-1 AACT. Discussion

[324] In this example several candidate biomarkers of toxic response to therapeutic or study agents, including compound PKI166, were determined. Some of these proteins, including the 43.4 kPa protein identified in this phase, demonstrated the potential to predict which patients are likely to develop grade 3 hepatotoxicity, even prior to treatment. Other candidate biomarkers, including the 11.7 kPa protein identified here, changed over the course of treatment with a peak level that corresponded to the time of peak toxicity. Unique purification schemes were developed for both the 43.4 kPa and 11.7 kPa proteins using a combination of column chromatography, size filtration and gel electrophoresis. At each stage of purification, the presence and purity of the proteins of interest were monitored on ProteinChip Arrays.

[325] Once the proteins were sufficiently purified, they were isolated on a SPS-PAGE gel, the bands containing the proteins of interest were excised, and the target proteins digested with proteases. Peptide maps were used to search the NCBI and SwissProt protein databases and positively identify the proteins of interest. The identification was confirmed by subjecting several peptides to MS/MS analysis to generate sequence information. [326] SAA. The 11.7 kPa protein was identified as SAA. SAA is a plasma apolipoprotein associated with the high density lipoproteins. It is synthesized by the liver and the proteolytic product has been shown to be an acute phase reactant. It has also been shown to be induced by cytokine stimulation. Processed SAA tends to aggregate into fibrils, resulting in complications associated with amyloidosis. The ability of SAA to form insoluble fibrils is interesting in light of its behavior in the profiling assay. The 11.5 kPa and 11.7 kPa peaks identified as SAA were present in both fraction 3 (pH 5) and fraction 6 (organic eluate). While there was a statistically significant change in the level of the 11.7 kDa protein which eluted from the anion exchange resin at pH 5, there was no significant change in the 11.7 kDa peak in fraction 6, which represents the aggregated form. In addition, while the 11.5 kDa peak was also identified as SAA, it did not exhibit a statistically significant change in patients that developed grade 3 hepatotoxicity.

[327] α-1 AACT. The 43.4 kDa protein was identified as α-1 AACT. AACT is a plasma protease inhibitor and is a member of the serine protease inhibitor class. It is synthesized in the liver and is an acute phase reactant, exhibiting increased levels in the plasma in response to trauma and infection. Low levels of AACT have been associated with liver disease. See Erikson et al. (1986), supra. Patients that developed grade 3 hepatotoxicity after treatment with PKI166 had lower levels of AACT prior to and during treatment. This suggests that these patients may have had low levels of liver disease that were not detected by measuring the AST levels.

[328] AACT is synthesized as a 433 amino acid precursor, the N-terminal 23 amino acids of which comprise a signal sequence. The mature form of AACT has a predicted mass of 45.3 kDa. It also contains several possible glycosylation sites. Glycosylation leads to the broadening of peaks detected by SELDI-TOF-MS. The AACT peak observed in this study is not unusually broad, suggesting an absence or a low level of glycosylation. The AACT detected in this study has a smaller mass (43.4 kDa) than the predicted mass and may indicate that the protein is posttranslationally processed. The N-terminal peptide was detected after trypsin digestion but the C-terminal peptide was not, suggesting the truncation may occur at the C-terminus.

List of Abbreviations for Example Avg M: Average Mass CHCA: a-Cyano-4-hydroxycinnamic acid CID: collision induced dissociation EAM: Energy Absorbing Molecule H4 or H50: Hydrophobic surfaces ID: identification I MAC: Immobilized metal affinity chromatography LC: Liquid chromatography Mono M: Monoisotopic Mass MS: Mass Spectrometry MS/MS: Mass spectrometry/mass spectrometry (tandem MS) MW: Molecular weight ACN: acetonitrile NP: Normal Phase PBS (buffer): Phosphate Buffer Saline PBSII: ProteinChip Biology System II pi: Isoelectric point Protein ID: Protein identification RC-MS: Retentate Chromatography - Mass Spectrometry SAX: Strong anion exchange SDS: Sodium dodecyl sulfate SELDI: Surface-Enhanced Laser Desorption/ionization SPA: Sinapinic acid TFA: Trifluoroacetic Acid TOF: Time-of-Flight WCX: Weak cation exchange Definitions Term Meaning Analyte Any atom and/or molecule; including their complexes and fragment ions. In the case of biological macromolecules including, but not limited to, protein, peptides, DNA, RNA, carbohydrates, steroids and lipids. Note that most important biomolecules under investigation for their involvement in the structure or regulation of life processes are quite large (typically several thousand times larger than H₂0). Molecular Ions Molecules in the charged or ionized state, typically by the addition or loss of one or more protons (H⁺). Molecular Fragmentation Breakdown products of analyte molecules caused, e.g., during laser- or Fragment Ions induced desorption, especially in the absence of added matrix Solid Phase The condition of being in the solid state, e.g., on the probe element surface. Gas or Vapour Phase Molecules in the gaseous state, i.e., in vacua for MS. Analyte Desorption/ The transition of analytes from the solid phase to the gas phase as ions. Ionization Note that the successful desorption/ionization of large, intact molecular ions by laser desorption is relatively recent (circa 1988)- the big breakthrough was the chance discovery of an appropriate matrix (nicotinic acid). Gas Phase Molecular Those ions that enter into the gas phase. Note: large molecular mass Ions ions, such as proteins (typical mass = 60,000-70,000 times the mass of a single proton) are typically not volatile, i.e., they do not normally enter into the gas or vapour phase. However, in the procedure of the present invention, large molecular mass ions such as proteins do enter the gas or vapour phase. Matrix In the case of MALDI, any one of several small, acidic, light absorbing chemicals, e.g., nicotinic or sinapinic acid, that is mixed in solution with the analyte in such a manner so that, upon drying on the probe element, the crystalline matrix-embedded analyte molecules are successfully desorbed (by laser irradiation) and ionized from the solid phase (crystals) into the gaseous or vapour phase and accelerated as intact molecular ions. For the MALDI process to be successful, analyte is mixed with a freshly prepared solution of the chemical matrix, e.g., 10,000:1 matrix:analyte, and placed on the inert probe element surface to air dry just before the MD analysis. The large fold molar excess of matrix, present at concentrations near saturation, facilitates crystal formation and entrapment of analyte. Energy Absorbing Any one of several small, light absorbing chemicals that, when Molecules (EAM) presented on the surface of a probe element (as in the case of SEND), facilitate the neat desorption of molecules from the solid phase, i.e., surface, into the gaseous or vapour phase for subsequent acceleration as intact molecular ions. The term EAM is preferred, especially in reference to SEND. Note that analyte desorption by the SEND process is defined as a surface-dependent process, I.e., neat analyte is placed on a surface composed of bound EAM. In contrast, MALDI is presently thought to facilitate analyte desorption by a volcanic eruption-type process that "throws" the entire surface into the gas phase. Furthermore, note that some EAM when used as free chemicals to embed analyte molecules as described for the MALDI process will not work, i.e., they do not promote molecular desorption, thus they are not suitable matrix molecules. Term Meaning

Probe Element or Sample An element having the following properties: it is inert, e.g., typically Presenting Device stainless steel; and active (probe elements with surfaces enhanced to contain EAM and/or molecular capture devices).

MALDI Matrix-Assisted Laser Desorption/ionization

TOF Time-of-Flight

MS Mass Spectrometry

MALDI-TOF-MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

ESI Electrospray ionization

Chemical Bonds Used simply as an attempt to distinguish a rational, deliberate, and knowledgeable manipulation of known classes of chemical interactions from the poorly defined kind of general adherence observed when one chemical substance, e.g., matrix, is placed on another substance, e.g., an inert probe element surface. Types of defined chemical bonds include electrostatic or ionic (+/-) bonds, e.g., between a positively and negatively charged groups on a protein surface, covalent bonds (very strong or "permanent" bonds resulting from true electron sharing), coordinate covalent bonds, e.g., between electron donor groups in proteins and transition metal ions, such as copper or iron; and hydrophobic interactions, such as between two non-charged groups.

Electron Donor Groups The case of biochemistry, where atoms in biomolecules, e.g., N, S and O, "donate" or share electrons with electron poor group, e.g., Cu ions and other transition metal ions.

Term Meaning

Allele An alternative form of a nucleotide sequence in a chromosome. Reference to an "allele" includes a nucleotide sequence in a gene or a portion thereof, as well as a nucleotide sequence that is not a gene sequence. Alleles occupy the same locus or position on homologous chromosomes. A subject having two identical alleles of a gene is considered "homozygous" for the allele, whereas a subject having two different alleles is considered "heterozygous". Alleles of a specific nucleotide sequence, e.g., of a gene can differ from each other in a single nucleotide, or several nucleotides, where the difference can be due to a substitution, deletion or insertion of one or more nucleotides. A form of a gene containing a mutation is an example of an allele. In comparison, a wild-type allele is an allele that, when present in two copies in a subject, results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.

Allelic Variant A portion of an allele containing a polymorphic region in the chromosomal nucleic acid. allelic variant of a A region of a gene having one of several nucleotide sequences found in polymorphic region of a that region of the gene in different individuals. gene

Biological sample Any material obtained from a living source, e.g., an animal, such as a human or other mammal, a plant, a bacterium, a fungus, a protist or a virus. The biological sample can be in any form, including a solid material, such as a tissue, cells, a cell pellet, a cell extract or a biopsy, or a biological fluid, such as urine, blood, saliva, amniotic fluid, exudate from a region of infection or inflammation, or a mouth wash containing buccal cells.

Term Meaning

Conditioned or When used in reference to a polypeptide, particularly a target Conditioning polypeptide, means that the polypeptide is modified so as to decrease the laser energy required to volatilize the polypeptide, to minimize the likelihood of fragmentation of the polypeptide, or to increase the resolution of a MS of the polypeptide or of the component amino acids. Resolution of a MS of a target polypeptide can be increased by conditioning the polypeptide prior to performing MS. Conditioning can be performed at any stage prior to MS and, in particular, can be performed while the polypeptide is immobilized. A polypeptide can be conditioned, e.g., by treating the polypeptide with a cation exchange material or an anion exchange material, which can reduce the charge heterogeneity of the polypeptide, thereby for eliminating peak broadening due to heterogeneity in the number of cations (or anions) bound to the various polypeptides in a population. Contacting a polypeptide with an alkylating agent, such as alkyliodide, iodoacetamide, iodoethanol or 2,3-epoxy-1-propanol, the formation of disulfide bonds, e.g., in a polypeptide can be prevented. Likewise, charged amino acid side chains can be converted to uncharged derivatives employing trialkylsilyl chlorides. Conditioning of proteins is generally unnecessary because proteins are relatively stable under acidic, high energy conditions so that proteins do not require conditioning for MS analyses. There are means of improving resolution, however, particularly for shorter peptides, such as by incorporating modified amino acids that are more basic than the corresponding unmodified residues. Such modification in general increases the stability of the polypeptide during MS analysis. Also, cation exchange chromatography, as well as general washing and purification procedures which remove proteins and other reaction mixture components away from the target polypeptide, can be used to clean up the peptide after in vitro translation and thereby increase the resolution of the spectrum resulting from MS analysis of the target polypeptide.

Delayed Extraction Methods in which conditions are selected to permit a longer optimum extraction delay and hence a longer residence time, which results in increased resolution (see, e.g., Juhasz ef al. (1996), supra; and Vestal et al., Rapid Commun Mass Spectrom, Vol. 9, pp. 1044-1050 (1995); see also, e.g., U.S. Patent Nos. 5,777,325; 5,742,049; 5,654,545; 5,641,959; 5, 654,545 and 5,760,393 for descriptions of MALDI and delayed extraction protocols). In particular, delayed ion extraction is a technique whereby a time delay is introduced between the formation of the ions and the application of the accelerating field. During the time lag, the ions move to new positions according to their initial velocities. By properly choosing the delay time and the electric fields in the acceleration region, the time of flight of the ions can be adjusted so as to render the flight time independent of the initial velocity to the first order. For example, a particular method involves exposure of the target polypeptide sample to an electric field before and during the ionization process, which results in a reduction of background signal due to the matrix, induces fast fragmentation and controls the transfer of energy prior to ion extraction. Term Meaning

Determining the identity Determining at least one characteristic of the polypeptide, e.g., the of a target polypeptide molecular mass or charge, or the identity of at least one amino acid, or identifying a particular pattern of peptide fragments of the target polypeptide. Determining the identity of a target polypeptide can be performed, e.g., by using MS to determine the amino acid sequence of at least a portion of the polypeptide, or to determine the pattern of peptide fragments of the target polypeptide produced, e.g., by treatment of the polypeptide with one or more endopeptidases. In determining the identity of a target polypeptide, the number of nucleotide repeats encoding the target polypeptide can be quantified.

Determining the identity The determination of the nucleotide sequence or encoded amino acid of an allelic variant of a sequence of a polymorphic region, thereby determining to which of the polymorphic region possible allelic variants of a polymorphic region that particular allelic variant corresponds.

In Vitro Transcription A cell-free system containing an RNA polymerase and other factors. System

Multiplexing Simultaneously determining the identity of at least two target polypeptides by MS. For example, where a population of different target polypeptides are present in an array on a microchip or are present on another type of solid support, multiplexing can be used to determine the identity of a plurality of target polypeptides. Multiplexing can be performed, e.g., by differentially mass modifying each different polypeptide of interest, then using MS to determine the identity of each different polypeptide. Multiplexing provides the advantage that a plurality of target polypeptides can be identified in as few as a single mass spectrum, as compared to having to perform a separate MS analysis for each individual target polypeptide.

Nucleotide Repeats Any nucleotide sequence containing tandemly repeated nucleotides. Such tandemly repeated nucleotides can be, e.g., tandemly repeated dinucleotide, trinucleotide, tetranucleotide or pentanucleotide sequences or any tandem array of repeated units.

Plurality In reference to a polynucleotide or to a polypeptide, means two or more polynucleotides or polypeptides, each of which has a different nucleotide or amino acid sequence, respectively. Such a difference can be due to a naturally-occurring variation among the sequences, for example, to an allelic variation in a nucleotide or an encoded amino acid, or can be due to the introduction of particular modifications into various sequences, e.g., the differential incorporation of mass modified amino acids into each polypeptide in a plurality.

Polymorphism The co-existence, in a population, of more than one form of an allele. A polymorphism can occur in a region of a chromosome not associated with a gene or can occur, e.g., as an allelic variant or a portion thereof of a gene. A portion of a gene that exists in at least two different forms, e.g., two different nucleotide sequences, is referred to as a "polymorphic region of a gene". A polymorphic region of a gene can be localized to a single nucleotide, the identity of which differs in different alleles, or can be several nucleotides long. Term Meaning Polypeptide At least two amino acids, or amino acid derivatives, including mass modified amino acids, that are linked by a peptide bond, which can be a modified peptide bond. A polypeptide can be translated from a nucleotide sequence that is at least a portion of a coding sequence, or from a nucleotide sequence that is not naturally translated due, for example, to its being in a reading frame other than the coding frame or to its being an intron sequence, a 3' or 5' untranslated sequence, or a regulatory sequence such as a promoter. A polypeptide also can be chemically synthesized and can be modified by chemical or enzymatic methods following translation or chemical synthesis. Protein, Polypeptide and Are used interchangeably herein when referring to a translated nucleic Peptide acid, e.g., a gene product. Quantify When used in reference to nucleotide repeats encoding a target polypeptide, means a determination of the exact number of nucleotide repeats present in the nucleotide sequence encoding the target polypeptide. As disclosed herein, the number of nucleotide repeats, e.g., trinucleotide repeats, can be quantified by using MS to determine the number of amino acids, which are encoded by the repeat, that are present in the target polypeptide. It is recognized, however, that the number of nucleotide repeats encoding a target polypeptide need not be quantified to determine the identity of a target polypeptide, since a measure of the relative number of amino acids encoded by a region of nucleotide repeats also can be used to determine the identity of the target polypeptide by comparing the mass spectrum of the target polypeptide with that of a corresponding known polypeptide. Reference polypeptide A polypeptide to which the target polypeptide is compared in order to identify the polypeptide in methods that do not involve sequencing the polypeptide. Reference polypeptides typically are known polypeptides.

References cited

[329] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

[330] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Ail patents, applications and publications referred to herein are incorporated by reference. For convenience, the meaning of certain terms and phrases used in the specification and claims are provided. [331] In addition, all GenBank accession numbers, Unigene Cluster numbers and protein accession numbers cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each such number was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. [332] The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatus within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

CLAIMSWe claim:

1. Use of ( )-4-[(1 -phenylethyl)amino]-7/- -pyrrolo[2,3-c]pyrimidin-6-yl]-phenol in the manufacture of a medicament for the treatment of proliferative disease with a reduced occurrence of liver toxicity, lung toxicity or both in a selected patient population, wherein the patient population is selected on the basis of the gene expression profile of the patients, wherein the gene expression profile comprises the gene expression products of one or more biomarker genes that are predictive of the occurrence of liver toxicity, lung toxicity or both in a patient following administration of (f?)-4-[(1-phenylethyl)amino]-7/-/-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol, wherein the one or more biomarker genes are selected from the group consisting of the SERPINA3 gene and the serum amyloid A (SAA) gene.

2. The use of claim 1 , wherein the gene expression product is a polynucleotide gene expression product.

3. The use of claim 1 , wherein the gene expression product is a polypeptide gene expression product.

4. A method for determining, prior to treatment, which individuals will develop dose- independent hepatotoxicity, lung toxicity or both when treated with a therapeutic or study agent; comprising: a) obtaining a sample of tissue or body fluid from the said individual prior to treatment; b) determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid to obtain a first value; c) determining the average value and the standard deviation in the values of the production of the said gene expression product of the SERPINA3 gene in at least 10 similar samples of tissue or body fluid from at least ten similar individuals to obtain a second value and standard deviation; d) comparing the first value with the second value; e) determining that the said individual is in a high risk group for developing dose-independent hepatotoxicity if the first value, determined in (a) is more than two standard deviations below the second value determined in (b); and f) determining that the said individual is in a low risk group if the first value determined in (a) is less than two standard deviations below the second value determined in (b) or is equal to or greater than the second value determined in (b).

5. The method of Claim 4, wherein the said sample of tissue or body fluid is selected from the group consisting of a tissue biopsy, blood, serum, plasma, lymph, ascitic fluid, cystic fluid, urine, cerebro-spinal fluid (CSF), salvia or sweat.

6. The method of Claims 4 or 5, wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the polypeptide expression product of the SERPINA3 gene in the said sample of tissue or body fluid.

7. The method of Claim 6, wherein the said polypeptide expression product of the SERPINA3 gene is the protein alpha-1 anti-chymotrypsin.

8. The method of Claims 4, 5, 6 or 7, wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid is preformed by measuring the level of the polypeptide gene expression product by means of mass spectrometry.

9. The method of Claim 8 wherein the mass spectrometry technique used is Surfaces Enhanced for Laser Desorbtion/lonization Time Of Flight Mass Spectrometry (SELDI- TOF-MS).

10. The method of Claim 9 wherein the mass spectrometry technique used is Matrix- Assisted Laser Desorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLDI- TOF-MS).

11. The method of Claim 6, wherein the presence of the polypeptide expression product of the SERPINA3 gene protein is detected using a reagent which specifically binds with the said polypeptide.

12. The method of Claim 11 , wherein the reagent is a labelled probe specific for the protein.

13. The method of Claim 11 , wherein the reagent is selected from the group consisting of an antibody, an antibody derivative and an antibody fragment.

14. The method of Claim 13, wherein the reagent is a monoclonal antibody.

15. A test kit for use in determining which individuals will develop dose-independent hepatotoxicity, lung toxicity or both when treated with a therapeutic or study agent; comprising the reagent of Claims 11 , 12,13 or 14 in a container suitable for contacting the said body fluid, with instructions for interpreting the results.

16. The test kit of Claim 15, wherein the reagent comprises an antibody, and wherein said antibody specifically binds with the polypeptide expression product of the SERPINA3 gene.

17. The method of Claim 4, wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the mRNA expression product of the SERPINA3 gene in the said sample of tissue or body fluid.

18. The method of Claim 17, wherein the level of expression of the mRNA expression product of the SERPINA3 gene is determined by techniques selected from the group consisting of: hybridization to a nucleotide array, Northern blot analysis, RT-PCR and real time quantitative PCR.

19. The methods of Claims 4-18 wherein the therapeutic or study agent is an epidermal growth factor receptor inhibitor (EGFRI).

20. The methods of Claims 4-19 wherein the epidermal growth factor receptor inhibitor (EGFRIs) is (R)-4-[(1-phenylethyl)amino]-7-/-pyrrolo[2,3-o pyrimidin-6-yl]-phenol.

21. The methods of Claims 4-18 wherein the therapeutic agent is an oxidizing drug.

22. A method for monitoring the progression or development of hepatotoxicity, lung toxicity or both in an individual being treated with a therapeutic or study agent, the method comprising: a) obtaining a pre-treatment sample of body fluid or tissue from the individual prior to administration of the agent; b) detecting a level of expression of the protein serum amyloid A (SAA) in the said body fluid or tissue sample; c) obtaining one or more post-administration samples of body fluid or tissue from the subject during or following treatment with the said therapeutic agent; d) detecting a level of expression of the protein SAA in one or more post- administration sample or samples; e) comparing the level of expression of protein SAA detected in (b) to the level detected in (d); and f) determining from the comparison of the two or more levels of SAA protein the likelihood that the individual is developing hepatotoxicity and adjusting the administration of the agent accordingly.

23. The method of Claim 22, wherein the said sample of tissue or body fluid is selected from the group consisting of; a tissue biopsy, blood, serum, plasma, lymph, ascitic fluid, cystic fluid, urine, cerbro-spinal fluid (CSF), salvia or sweat.

24. The method of Claim 22 or 23, wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the mRNA expression product of the SERPINA3 gene in the said sample of tissue or body fluid.

25. The method of Claim 24, wherein the level of expression of the mRNA expression product of the SERPINA3 gene is determined by techniques selected from the group consisting of Northern blot analysis, RT-PCR and real time quantitative PCR.

26. A method for monitoring the progression or development of hepatotoxicity, lung toxicity or both in a subject having, or at risk of having, hepatotoxicity, lung toxicity or both during or after treatment with a therapeutic or study agent, comprising measuring a level of expression of the gene expression product of the SAA gene over time in a sample of bodily fluid or tissue obtained from the subject during treatment, wherein an increase in the level of expression of the said protein over time is indicative of the development of hepatotoxicity, lung toxicity or both in the subject.

27. The method of Claim 26 wherein the step of measuring the level of production of the gene expression product of the SAA gene protein SAA over time is done by measuring the level of SAA protein in a sample of bodily fluid or tissue obtained from the subject.

28. The method of Claim 26 or 27 wherein the step of determining the level of expression of the protein SAA in the said sample of tissue or body fluid is preformed by measuring the level of the protein SAA by means of mass spectrometry.

29. The method of Claim 28 wherein the mass spectrometry technique used is Surfaces Enhanced for Laser Desorbtion/lonization Time Of Flight Mass Spectrometry (SELPI- TOF-MS)

30. The method of Claim 28 wherein the mass spectrometry technique used is Matrix- Assisted Laser Pesorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLPI- TOF-MS).

31. The method of Claim 26 or 27, wherein the presence of the protein SAA is detected using a reagent which specifically binds with the said polypeptide.

32. The method of Claim 31 , wherein the reagent is selected from the group consisting of an antibody, an antibody derivative and an antibody fragment.

33. A test kit for use in determining which individuals are developing dose-independent hepatotoxicity, lung toxicity or both when treated with a therapeutic or study agent; comprising the reagent of Claims 31 or 32 in a container suitable for contacting the said body fluid with instructions for interpreting the results.

34. The test kit of Claim 33, wherein the reagent comprises an antibody, and wherein said antibody specifically binds with the protein SAA.

35. The method of Claim 26, wherein the step of determining the level of production of the gene expression product of the SAA gene in the said sample of tissue or body fluid is performed by determining the level of the mRNA expression product of the SAA gene in the said sample of tissue or body fluid.

36. The method of Claim 35, wherein the level of expression of the mRNA expression product of the SAA gene is determined by techniques selected from the group consisting of: hybridization to a nucleotide array, Northern blot analysis, RT-PCR and real time quantitative PCR.

37. The methods of Claims 26-36, wherein the therapeutic agent is an epidermal growth factor receptor inhibitor (EGFRI).

38. The method of Claim 37 wherein the epidermal growth factor receptor inhibitor (EGFRI) is (R)-4-[(1 -phenylethyl)amino]-7/--pyrrolo[2,3-o]pyrimidin-6-yl]-phenol.

39. The methods of Claims 26-36, wherein the therapeutic agent is an oxidizing drug.

40 A method for determining, prior to initiation of treatment, which individual(s) should be included in a study of a therapeutic or study agent; comprising: a) obtaining a sample of tissue or body fluid from the said individual(s) prior to treatment; b) determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid to obtain a first value; c) determining the average value and the standard deviation in the values of the production of the said gene expression product of the SERPINA3 gene in at least 10 similar samples of tissue or body fluid from at least ten similar individuals to obtain a second value and standard deviation; d) comparing the first value with the second value; e) determining that the said individual is in a high risk group for developing dose-independent hepatotoxicity, lung toxicity or both, and should not be included in the treatment or study, if the first value, determined in (a) is more than two standard deviations below the second value determined in (b); and f) determining that the said individual is in a low risk group and may be included in the treatment or study, if the first value determined in (a) is less than two standard deviations below the second value determined in (b) or is equal to or greater than the second value determined in (b).

41. The method of Claim 40, wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid prior to treatment is performed by determining the level of the polypeptide expression product of the SERPINA3 gene in the said sample of tissue or body fluid.

42. The method of Claims 40 or 41 , wherein the said sample of tissue or body fluid is selected from the group consisting of a tissue biopsy, blood, serum, plasma, lymph, ascitic fluid, cystic fluid, urine, cerebro-spinal fluid (CSF), salvia or sweat.

43. The method of Claim 41 , wherein the said polypeptide expression product of the SERPINA3 gene is the protein alpha-1 anti-chymotrypsin.

44. The method of Claims 40-43 wherein the step of determining the level of production of the gene expression product of the SERPINA3 gene in the said sample of tissue or body fluid is preformed by measuring the level of the polypeptide gene expression product by means of mass spectrometry.

45. The method of Claim 44 wherein the mass spectrometry technique used is Surfaces Enhanced for Laser Pesorbtion/lonization Time Of Flight Mass Spectrometry (SELDI- TOF-MS).

46. The method of Claim 44 wherein the mass spectrometry technique used is Matrix- Assisted Laser Desorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLDI- TOF-MS).

47. The method of Claims 40-43, wherein the presence of the polypeptide expression product of the SERPINA3 gene protein is detected using a reagent which specifically binds with the said polypeptide.

48. The method of Claim 47, wherein the reagent is a labelled probe specific for the protein.

49. The method of Claims 47 or 48, wherein the reagent is selected from the group consisting of an antibody, an antibody derivative and an antibody fragment.

50. The method of Claim 49, wherein the reagent is a monoclonal antibody.

51. A test kit for use in determining which individuals will develop dose-independent hepatotoxicity, lung toxicity or both when treated with a therapeutic or study agent and should not be included in a study of that therapeutic agent; comprising the reagent of Claim 47-50, in a container suitable for contacting the said body fluid with instructions for interpreting the results.

52. The test kit of Claim 51 , wherein the reagent comprises an antibody, and wherein said antibody specifically binds with the polypeptide expression product of the SERPINA3 gene.

53. A method for determination of when treatment with a therapeutic or study agent should be discontinued in a subject at risk of having, hepatotoxicity, lung toxicity or both during or after treatment with a therapeutic or study agent, comprising measuring a level of expression of the protein SAA over time in a sample of bodily fluid or tissue obtained from the subject during treatment, wherein an increase in the level of expression of the said protein over time is indicative of the development of hepatotoxicity, lung toxicity or both in the subject and determines that the agent should be discontinued.

54. The method of Claim 53 wherein the step of determining the level of expression of the protein SAA in the said sample of tissue or body fluid is preformed by measuring the level of the protein SAA by means of mass spectrometry.

55. The method of Claim 54 wherein the mass spectrometry technique used is Surfaces Enhanced for Laser Desorbtion/lonization Time Of Flight Mass Spectrometry (SELDI- TOF-MS).

56. The method of Claim 54 wherein the mass spectrometry technique used is Matrix- Assisted Laser Desorbtion/lonization, Time Of Flight, Mass Spectrometry (MASLDI- TOF-MS).

57. The method of Claim 53, wherein the presence of the protein SAA is detected using a reagent which specifically binds with the said polypeptide.

58. The method of Claim 57, wherein the reagent is selected from the group consisting of an antibody, an antibody derivative and an antibody fragment.

59. The method of Claim 58, wherein the reagent is a monoclonal antibody.

60. A test kit for use in determining when a therapeutic or study agent should be discontinued for an individual being treated with a therapeutic or study agent; comprising the reagent of Claim 57, 58 or 59, in a container suitable for contacting the said body fluid with instructions for interpreting the results.

61. The test kit of Claim 60, wherein the reagent comprises an antibody, and wherein said antibody specifically binds with the protein serum amyloid A (SAA).

62. The method of Claim 53, wherein the step of determining the level of production of the gene expression product of the SAA gene in the said sample of tissue or body fluid is performed by determining the level of the mRNA expression product of the SAA gene in the said sample of tissue or body fluid.

63. The method of Claim 62, wherein the level of expression of the mRNA expression product of the SAA gene is determined by techniques selected from the group consisting of: hybridization to a nucleotide array, Northern blot analysis, RT-PCR and real time quantitative PCR.

64. The method of Claim 53 wherein the therapeutic agent is an epidermal growth factor receptor inhibitor (EGFRI).

65. The method of Claim 64 wherein the epidermal growth factor receptor inhibitor (EGFRI) is (R)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-o]pyrimidin-6-yl]-phenol.

66. The method of Claim 53 wherein the therapeutic agent is an oxidizing drug.