WO2006122424A1 - Protein depletion using volatile binding buffers - Google Patents

Abstract

Methods have been developed for purifying a sample by protein depletion that comprise a step of incorporating a volatile binding buffer into a biological sample prior to protein depletion The depleted sample can then be desalted in situ by vacuum evaporation, thereby increasing sample compatibility with post-depletion processing techniques while maintaining sample integrity, as compared with more labour intensive desalting methods such as dialysis, ultrafiltration, and size-exclusion The methods are useful in applications requiring protein or peptide depletion techniques, such as lmmuno-affimty depletion in sample processing for medical diagnostics, industrial purification, or proteomics

Description

PROTEIN DEPLETION USING VOLATILE BINDING BUFFERS

Field of the Invention

The invention relates to protein purification and analysis,

Background Removal or depletion of high abundance proteins is a common approach in sample preparation for proteomics studies of blood plasma. Immuno-affinity (IA) depletion is currently an accepted method for performing this step. Major manufacturers of immunoafϊinity (IA) depletion matrices (e.g. Agilent Technologies, Amersham Biosciences, and Gen Way) usually provide chromatography protocols based on non- volatile binding buffers, though the use of volatile buffers (acetic acid, for example) for elution in affinity chromatography methods is widely accepted. The components of these binding buffers (inorganic ions) frequently have limited compatibility with downstream steps of sample processing (enzymatic digestion, ion- exchange chromatography, etc.). Therefore, they are generally removed or exchanged prior to further sample processing. The majority of salt exchange or removal methods have near quantitative sample recovery (Liao et al. (2004) Arthritis Rheum. 50: 3792; Varnerin et al, (1998) Protein Express. Purif. 14: 335) for moderately soluble proteins at relatively high concentrations. However, the losses dramatically increase for samples composed of proteins with low solubility (West el al. (1998) Biotechnol. Bioeng. 57: 590). This negative impact on recovery can also be exacerbated if the sample or its key constituents are of limited quantity at the outset. Moreover, salt exchange methods require additional labor and materials, increasing the processing time and cost of analysis. Brief Summary of the Invention

The inventors have discovered that volatile buffers, generally used for elution steps in immunoaffinity applications, can be used as a binding buffer in immunoaffinity applications. The invention features methods and kits that include the use of volatile buffers for sample preparation or as binding buffers in immunoaffinity applications, such as immunoaffinity depiction. The use of volatile buffers as sample preparation buffers or binding buffers provides several advantages including improved recovery of proteins from samples subjected to immunodepletion and increased efficiency of immunodepletion. Immunodepletion methods that incorporate the use of a volatile binding buffer overcome the disadvantages associated with desalting (and resulting sample toss) of samples solubilized in nonvolatile buffers by allowing for a depleted plasma sample to be desalted "in-situ" using vacuum evaporation techniques, such as freeze drying. This method has been successfully implemented by the inventors into sample processing for mass spectrometric proteomic analysis of blood plasma, for which application an improvement over the nonvolatile buffer method has been demonstrated. The invention accordingly relates to methods of using volatile binding buffers in affinity-based separations based on covalent or non-covalent mechanisms of interactions between capture reagents and target molecules (e.g. cysteine-based depletion or immobilized metal ion chromatography).

The present invention, in genera], provides a method for purifying a sample including one or more proteins by incorporating a volatile buffer into the sample, contacting the sample with one or more affinity reagents, and separating the portion of the sample not specifically bound to the affinity reagent(s), thereby depleting the sample. In one embodiment, the purified sample is further subjected to vacuum evaporation. The purified and vacuum evaporated sample may also be further subjected to analysis by mass spectrometry.

In certain embodiments of the invention, the volatile buffer contains ammonium ions, such as ammonium formate, ammonium acetate, or ammonium bicarbonate. For example, ammonium bicarbonate may be present in a sample in a concentration of about 50 mM to 250 mM (e.g., about 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM). In another embodiment, the proteins have low solubility. In still another embodiment, the sample is less than 0.1 microgram per microliter in concentration. In yet another embodiment, the sample is cellular material, blood plasma, cerebrospinal fluid, saliva, and urine or cell lysate.

An affinity reagent described herein includes any affinity reagent that specifically binds one or more biomolecules, such as one or more proteins. Affinity reagents are typically attached to a solid support, such as a bead, a filter, a planar surface, or a well of a multi-well plate. Exemplary affinity reagents include affinity and immunoaffinity reagents or immunosubtractors.

In one embodiment, the method is used to compare the relative abundance of peptides in a sample, or population (e.g., 2, 3, 4, 5, 10, 20, 50, or more) of samples, as compared to the abundance in a reference, such as a reference sample or population (e.g., 2, 3, 4, S, 10, 20, 50, or more) of reference samples (e.g., a control sample). For this embodiment, the method further includes measuring the expression levels of one or more peptides in the sample and comparing the expression levels of the one or more peptides from the sample to the expression levels in the reference. Differences in the relative abundance of the peptides from the sample to the reference can be used to identify differentially expressed peptides. Such a method can be used, for example, to identify differentially expressed peptides in a sample having altered activity as compared to the reference. The method can also be used to screen for conditions that alter the expression levels of one or more peptides in the sample as compared to the reference,

In other aspects, the invention relates to protein separation methodologies such as those that involve centrifugation or chromatography. If desired, the methods disclosed herein are amenable to semi-automated or fully-automated techniques such as those involving robotics according to standard methods known in the art.

The invention also provides methods of screening proteins in a sample that are not specifically bound by an affinity reagent. This method includes incorporating a volatile buffer into the sample, contacting the sample with one or more affinity reagents, separating the portion of the sample not specifically bound to the affinity reagent(s), thereby purifying the sample, and identifying one or more of the non- binding proteins. In a particular embodiment of the screening method, one or more of the affinity reagents bind phosphorylated or glycosylated peptides, In another embodiment, the sample comprises a multi-subunit complex.

The invention also provides a kit that includes a volatile buffer and instructions for the use of the volatile buffer as a binding buffer, for example, for use in an immunoaffinity assay.

The compositions and methods of the invention are useful for improved recovery, and/or improved efficiency in recovery, of non-binding proteins from samples contacted with an affinity reagent, such as blood plasma samples subjected to immi∞odepletion. The methods described herein advantageously provide for improved protein recovery, speed of sample processing, and reproducibility of results, as well as lower cost of materials.

Other features and advantages will be apparent from the following description and the claims.

Brief Description of the Drawing

Figure 1 is a graph showing UV traces (280 run) of depletion runs in volatile and non-volatile conditions. This figure indicates that the use of volatile buffer neither affects the retention time nor the shape of the peak of depleted plasma proteins (unbound proteins). UV profiles (280 nm) of plasma depletion under volatile (solid line) and non-volatile (dotted line) conditions are illustrated. Unbound proteins elute at the same time under both conditions. Bound proteins elute slightly later in volatile conditions.

Detailed Description of the Invention Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated, as used herein, the following terms are intended to have the following meanings in interpreting the present invention. As used herein, "activity" includes one or more measurable properties of a protein, capable of acting on or affecting a change on itself, another molecule, or a cell, tissue, organ, or organism. Although "activity" may often be taken to imply active function, it is further meant to encompass measurable passive functions as well (e.g., maintaining structural confoπnarion of a particular protein complex), preferably those that relate to cancer or disease phenotypes or mechanisms. Some examples, not intended to be limiting, include catalytic enzymatic activity, translocation, binding, immunological activity (including specifically imtnunogenicity), or participation in a biochemical, or phenotypic pathway that are monitored and evaluated according to standard methods. The activity may be carried out indirectly, such as through functioning in a pathway, and encompasses activities that require co-factors or presence in a protein complex. A percentage activity can be determined by comparison to a control in an assay for the particular activity being examined. Methods for such comparisons are commonly known in the art. For example, the percent kinase activity of a derivative of a protein kinase can be assessed by comparison to the level of activity of underivatized protein kinase under appropriately similar conditions in a kinase assay. Activities may be self-directed, such as auto- catalytic activity. Some assays may require the use of protein encoding nucleic acids, such as for expression, or producing transgenic cell lines, or specific mutant, variant, or derivative forms of a protein being examined.

Some activity assays, not intended to be limiting, that may be useful in carrying out the methods of the invention, including identifying functions of polypeptides and nucleic acids include cell proliferation assays, such as mitotic index (see, for example, Oka et at. (1994) Arch Pathol Lab Med. 118: 506 - 509; Weidixer et al. (1994) Hum Pathol. 25: 337 - 342), thymidine incorporation assays (see, for example, Rodriguez et al. (1993) Am J Obstet Gynecol. 168: 228 - 232; Sugihara et al. (1992) Int J Cell Cloning 10: 344 - 351; Hayward et al (1992) Int J Cell Cloning 10: 182 - 189; Sondak et al. (1988) Int J Cell Cloning 6: 378 - 391), bromodeoxyuridine (BrdU) incorporation assays (see, for example, Limas (1993) J Pathol. 171 : 39 - 47), MIB-I staining (see, for example, Spyratos et al. (2002) Cancer 94: 2151 - 2159), or anti-PCNA (proliferating cell nuclear antigen) staining (see, for example, Hall et al. (1990) J Pathol. 162: 285 - 294; Kurki et at. (1988) J Immunol Methods 109: 49 - 59; Kubben et al (1994) Gut 35: 530 - 535; and the in situ hybridization method of Kohler et al (2004 Dec 23; Epub ahead of print) Histochem Cell Biol.); growth suppression assays, such as assays of susceptibility to arrest (see, for example, Guan et al. (1994) Genes Dev. 8: 2939 - 2952; Gulliya et al. (1994) Cancer 74: 1725 - 1732), and drug resistance assays (for example, Vybrant®

Multidrug Resistance Assay Kit, catalog # V13180 from Molecular Probes, Eugene, Oregon); apoptosis assays, such as DAPI staining, TUNEL assay (e.g., Fluorescein FragEL DNA Fragmentation Detection Kit (Oncogene Research Products, Cat.# QIA39)+Tetramethyl-rhodamine-5-dUTP (Roche, Cat. # 1534 378)) or APO-BrdU™ TUNEL Assay Kit, catalog # A23210 from Molecular Probes, Eugene, Oregon) or an assay based on Protease Activity (such as caspases) (for example, EnzChek® Caspase-3 Assay Kit #1, catalog # E13183 from Molecular Probes, Eugene, Oregon); angiogenesis assays (see, for example, Storgard et al. (2004) Methods MoI Biol. 294: 123 - 136; Baronikova et at. (2004) Planta Med. 70: 887 - 892; Hasan et al (2004) Angiogenesis 7: 1 - 16; Friis et al. (2003) APMIS. 111: 658 - 668); cell migration assays (for example, Yarrow et al. (2004) BMC Biotechnol. 4: 21; Berens and Beaudry (2004) Methods MoI Med. 88: 219 - 24; Heit and Kubes (2003) Sci STKE. 2003 (170): PL5); cell adhesion assays (for example, those using enzyme substrates, such as the Vybrant® Cell Adhesion Assay Kit, catalog # Vl 3181 from Molecular Probes, Eugene, Oregon); assays of ability to grow on soft agar or colony formation assays (see, for example, Freshney (1994) Culture of Animal Cells a Manual of Basic Technique. 3rd ed., Wiley-Liss, New York); assays for changes in contact inhibition or density limitation of growth (see, for example, Freshney (1994), supra); assays of changes in growth factor or serum dependence (see, e.g., Temin (1966) J Natl Cancer Insti.37: 167 - 175; Eagle et al. (1970) J Exp Med. 131: 836 - 879; Freshney (1994), supra); assays of changes in the level of tumor specific markers (for example, Mazumdar et al (1999) Trop Gastroenterol. 20: 107 - 110; Rosandic et al (1999) Acta Med Austriaca. 26: 89 - 92; Clarke et al (2003) Int J Oncol. 22: 425 - 30; Nowak et al. (2003) Eur J Gastroenterol Hepatol. 15: 75 - 80; Sarkar et al. (2002) Int J Pharrn. 238: 1 - 9; Streckfus et al (2001) Oral Surg Oral Med Oral Pathol Oral

Radiol Endod. 91: 174 - 179; Werther et al. (2000) Eur J Surg Oncol. 26: 657 - 662; Halberg et al. (1995) In Vivo. 9: 311 - 314; Varela et al (1993) Oncology 50: 430 - 435; Turner et al. (1990) Eur J Gynaecol Oncol. 11: 421 -427; Masood (1994) J Cell Biochem Suppl. 19.' 28 - 35; Vogel and Kalthoff (2001) Virchows Arch. 439: 109 - 117); assays of changes in invasiveness into Matrigel (see, for example, Freshney (1994), supra); assays of changes in cell cycle pattern (for example, as determined by flow cytometry, or mRNA or protein expression in synchronized cells (see, for example, Li et al. (1994) Oncogene 9: 2261 - 2268); assays of changes in tumor growth in vivo, such as in transgenic mice (for example, Huh et al. (2005) Oncogene 24: 790 - 800; White et al. (2004) Cancer Cell 6: 159 - 170; Finkle et al. (2004) Clin Cancer Res. 10: 2499 - 2511; Williams et al. (2004) J Biol Chem. 279: 24745- 24756; Cuadros et al. (2003) Cancer Res. 63: 5895 - 5901; Quaglino et al. (2002) Immunol Lett. 80: 75 - 79; Shibata et al. (2001) Cancer Gene Ther. 8: 23 - 35; Nielsen el al. (2000) Cancer Res. 60: 7066 - 7074), or in xenografts (for example, in immune suppressed mice, such as SCID mice; see Houghton et al. (1989) Invest New Drugs. 7: 59 - 69; Rygaard and Spang-Thomsen (1997) Breast Cancer Res Treat. 46: 303 - 312; van Weerden and Romijn (2000) Prostate 2000 43: 263 - 271 ; Azzoli el al. (2002) Semin Oncol. 29: 59 - 65; Sliwkowski et al. (1999) Semin Oncol. 26: 60 - 70); binding assays; known cancer diagnostics; etc. Such assays can be used to screen for anti-cancer agents, including identification of nucleic acids or polypeptides which are capable of altering or inhibiting abnormal proliferation and transformation in host cells, and activators, inhibitors, and modulators of nucleic acids and polypeptides. Such activators, inhibitors, and modulators are useful to modulate expression in tumor cells or abnormal proliferative cells. Identified nucleic acids or polypeptides that inhibit abnormal proliferation and transformation in host cells are useful in a number of diagnostic or therapeutic methods, e.g., in gene therapy to inhibit abnormal cellular proliferation and transformation. As used herein, "affinity" refers to strength of binding between substances. In one example, a high binding affinity is generally desired, for example, between an antibody and its antigen. In other examples, a specific and high affinity compound is used to purify a specific protein. In other examples, a low affinity compound is used, to isolate several members of a protein family. By "high binding affinity" is meant binding having an affinity constant of less than 1 micromolar, preferably, less than 100 nanomolar, and more preferably, 10 nanomolar to 1 picoroolar. Binding and affinity assays known in the ail: are used to determine affinity or to screen for high affinity binding compounds.

As used herein, "affinity reagent" or "capture reagent" is a substance that binds to a target molecule. Generally, such binding is specific. The affinity of such reagents may vary. In one example, the affinity is high enough to reasonably meet the aims of the method they are used to address and are typically of high binding affinity. However, a collection of low affinity binders can be combined to provide a high affinity equivalent (high avidity). High avidity/affinity reagents are also useful in the methods described herein. Such reagents are often used for their specificity in separation or purification methods. In some cases less discriminating reagents may be preferable, such as those that could effectively bind and deplete a family of proteins via a similar or common epitope, but in other cases highly discriminant or specific reagents capable of distinguishing even small differences between similar proteins may be preferred. An example of a capture reagent is nickel, such as may be present in a column to purify histidine-tagged proteins from a bacterial cell lysate.

Immunoaffinity reagents are affinity reagents composed at least in part of naturally occurring or engineered antibodies, antibody fragments, including CDR peptides, and the like. Immunoaffinity reagents may recognize one or more antigens or epitopes.

"Altered" or "changed" refers to a detectable change or difference from a reasonably comparable state, profile, measurement, or the like, One skilled in the art should be able to determine a reasonable measurable change. Such changes may be all or none. They may be incremental and need not be linear. They may be by orders of magnitude- A change may be an increase or decrease by 1%, 5%, 10%, 20% ,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more, or any value in between 0% and 100%.

As used herein, "antibody" refers to an immunoglobulin protein (or proteins such as in the case of a polyclonal antibody) whether naturally or synthetically produced, which is capable of binding an antigen that caused its production. The term may be used to encompass the antibody, antibody fragments, a polypeptide substantially encoded by at least one immunoglobulin gene or fragments of at least one immunoglobulin gene which can participate in specific binding with the antigen, naturally-occurring forms, conjugates, and derivatives thereof. The immunoglobulin molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD, or IgA) or subclass of immunoglobulin molecule. The term also covers any protein having a binding domain homologous to or derived from an immunoglobulin binding domain, such as a CDR region or a cyclized peptide based on a CDR amino acid sequence. Terms such as "antigen-binding region of an antibody" may also be used to encompass CDR regions and the like. An antibody can be derived from a sequence of a mammal, non-mammal (e.g., birds, chickens, fish, etc.), or fully synthetic antibody sequences. A "mammal" is a member of the class Mammalia. Examples of mammals include, without limitation, humans, primates, chimpanzees, rodents, mice, rats, rabbits, sheep, and cows.

Derivatives within the scope of the term include antibodies that have been modified in sequence but remain capable of specific binding to a target molecule including interspecies, chimeric, and humanized antibodies. When they are naturally produced, antibodies are secreted into the bloodstream to seek out and bind foreign agents or antigens for destruction. An antibody may be monoclonal or polyclonal and present in a variety of media including, but not limited to, serum or supernatant, or in purified form. As used herein, antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, hybridomas, recombinant expression systems, by phage display, or other methods known in the art. Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein or biomolecule, is mixed with an adjuvant and animals are immunized. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antiserum is prepared. Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler and Milstein (1976) Eur. J. Immunol. 6; 511 - 519). Other methods of immortalization include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.

As used herein, "antibody fragment" or "antibody protein fragment" refers to a portion of an antibody (i.e., Fv) capable of binding to an antigen. Fragments within the scope of the term as used herein include those produced by digestion with various peptidases, such as Fab, Fab' and F(ab)'2 fragments, those produced by chemical dissociation, by chemical cleavage, and recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Typical recombinant fragments (e.g., those produced using phage display technology) include single chain Fab and scFv ("single chain variable region") fragments. Derivatives within the scope of the term include those that have been modified in sequence, but remain capable of specific binding to a target molecule, including interspecies, chimeric, and humanized antibodies.

As used herein, "antigen" refers to a substance that is or will be introduced or injected into a vertebrate animal such as a mammal or poultry; or presented by antigen presentation machinery; or brought into contact with a T cell, B cell, or antigen presenting cell to induce an immune response, particularly the formation of specific antibodies that can combine or bind with the antigen. An antigen need not be immunogenic. Antigens that can induce an immune response are often referred to as immunogenic. Antigens, such as peptides, may be tested to determine immunogenicity by an appropriate assay, as may be known in the art. For example, an assay for immunogenicity is the production of antibodies that recognize the antigen, such as in immunoprecipitatioπ or immimoblotting, in response to antigen challenge. Another example is assaying for T cell stimulation by an antigen (e.g., polypeptide). T cells may be stimulated with a polypeptide, polynucleotide encoding a polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide of interest. An antigen, such as a polypeptide or polynucleotide, is typically present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells. T cells are considered to be activated by a polypeptide if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the antigen polypeptide or expressing a gene encoding the polypeptide. T cell activation may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two-fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al. (1994) Cancer Res. 54: 1065 - 1070. Detection of the proliferation of T cells may also be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Contact with an immunogenic polypeptide (100 ng/ml - 100 μg/ml, preferably 200 ng/ml - 25 μg/ml) for 3 - 7 days typically results in at least a two-fold increase in proliferation of the T cells. Such contact, as described above, for 2 - 3 hours typically results in activation of the T cells, as measured using standard cytokine assays in which a two-fold increase in the level of cytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al. (1998) Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells that have been activated in response to an immunogenic polypeptide, polynucleotide, or polypeptide- expressing APC may be CD4⁺ and/or CDS⁺). The immunogenicity of an antigen is also predicted using standard software programs known in the art. Antigens taken up and presented by an antigen presenting cell may be referred to herein as antigen presenting cell antigens. Antigens capable of activating T cells or B cells may be referred to herein as T cell antigens and B cell antigens, respectively. An adjuvant may accompany an antigen to provide an additional degree of immunogenicity. The portions of the antigen that make contact with the antibody are denominated "epitopes." Antigens can be derived from a broad range of sources and can include, for example, viruses, proteins, nucleic acids, and organic compounds. Encompassed within this term herein are haptens, small antigenic determinants capable of inducing an immune response only when coupled to a carrier. Haptens bind to antibodies but by themselves cannot induce an antibody response. As used herein, "binding" refers to a non-covalent or a covalent interaction, preferably non-covalent, that holds two molecules together. For example, two such molecules could be an enzyme and an inhibitor of that enzyme. Another example would be an enzyme and its substrate. A third example would be an antibody and an antigen. Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions among charged groups, van der Waals interactions, and hydrophobic interactions among non-polar groups. One or more of these interactions can mediate the binding of two molecules to each other. Binding, in some instances, exhibits discriminatory properties such as specificity or selectivity.

As used herein, "blood plasma" refers to the fluid portion of whole blood that consists of water and its dissolved constituents including proteins (such as albumin, fibrinogen, and globulins), electrolytes (such as sodium and chloride), sugars (such as glucose), lipids (such as cholesterol and triglycerides), metabolic waste products (such as urea), amino acids, hormones, and vitamins. Exemplary blood plasma useful in the methods described herein is mammalian blood plasma (e.g., human blood plasma).

As used herein, "cellular material" refers to a sample including cells and/or contents and components thereof. The term also encompasses a sample such as a tissue, organ, organism, or homogenate or fraction thereof and may contain some portion of extracellular material in addition to cells and/or the contents and components thereof.

As used herein, "centrifugation" refers to a method for separating substances of different densities using centripetal force (the physics of particles in suspension also allows separation of spherical particles of equal densities under a specific set of conditions with the variable that determines the force on a given particle being its radius). Generally a machine is used to subject a sample or samples to centrifugal action, such as in a microfuge, centrifuge, or ultracentrifuge, which generally have speeds between \ and 70,000 revolutions per minute (rpm). As used herein, "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the solutes as they flow around or over a stationary liquid or solid phase. Chromatography generally refers to liquid chromatography, chromatography in which the mobile phase is a liquid. Generally, as used herein, chromatography also refers to affinity chromatography or chromatography in which one or more macromolecules (such as a protein) is isolated and purified by passing it in solution through a column comprising one or more affinity reagents for the macromolecule, causing it to be retained on the column and removed from a sample. In immunoaffinity chromatography at least one affinity reagent is an immunoaffϊnity reagent. As used herein, "contacting" refers to the bringing together or combining of two or more substances, such as a plasma sample and an affinity reagent as embodied in a chromatography column, such that they are within a distance permitting, and present for a sufficient time for, the appropriate intermolecular interactions and/or chemical transformations to occur. Contacting preferably occurs with a sample in solution phase to permit free association of sample molecules with the substance it is brought into contact with. "Contacting" may be used interchangeably with terms like combined with, added to, mixed with, passed over, and incubated with.

As used herein, "functional assay" refers to assays to determine the degree to which abiomolecule, such as a protein, exhibits a particular activity. For example, a phenotypic assay may be used to determine the contribution to a particular phenotype, a kinase assay may be used to determine kinase activity and preferences for particular substrates. The "degree" may be all or none, an absolute measurable amount, a range of variation (e.g. with a pleiotropic phenotype), or an amount relative to another sample, such as a reference sample or control sample. "Genotype" refers to the genomic make-up of an organism, and two organisms with different genomic sequences or containing different genes or alleles thereof are considered of different genotypes for purposes of the invention (though they maybe phenotypically identical to an observer). So, for example, in murine genetics, the strain is generally referred to as the genetic background into which various transgenes are integrated, but for the purposes of the invention 10 C57BL/6J mice with different integrated transgenes would be considered different genotypes (in this case genotype referring to a difference in potentially just one or two genes), and potentially better scientific controls for each other than a more diverse sample population. Samples from a population of human patients are likely to have more diverse genotypes. As used herein, "identifying one or more of the proteins present" is not necessarily synonymous with determining the sequence and includes partially identifying the polypeptide or characterizing it as similar to or different from a known protein. Further, it includes making a tentative identification based on the most probable of a small number of possibilities, and maybe based wholly or in part on characteristics such as electrophoretic mobility, mass-to-charge ratio, peptide mass fingerprinting, molecular weight, isoelectric point, retention time in chromatography, proteolysis patterns (such as those produced by V8 protease mapping or mapping with N-chlorosuccinimide), immunoreactivity (e.g. binding with a specific antibody in an immunoassay such as an ELISA), binding characteristics, or other characteristics. For example, identification maybe by a combination of mass-to-charge ratio (m/z) and retention time or a corrected version thereof, such as is used to align peptides in U.S. Patent Application Publication No. 20040172200. Preferably identification based on such characterization is unambiguous, but need not be, particularly in preliminary efforts. For the purposes of identification "proteins" may be used to refer to relatively short peptides, but preferably refers to full length proteins, such as might be encoded by a cellular mKNfA, and that may require one or more peptides be identified to unambiguously assert that the whole protein is likely to have been present. The identification may be confirmatory based on prior knowledge or prediction. Exemplary methods of identification include mass spectrometry and tandem mass spectrometry with accompanying sequence determination. Sequence determination by tandem mass spectrometry or other methods may not always be complete (thus being only "in part"), but it may be sufficient to distinguish one protein from another or to uniquely identify the parent protein for a peptide, particularly if combined with other characteristics such as m/z and retention time. It may be desirable to identify the proteins in a sample as completely as possible, only a few, or possibly only one. It may also be desirable to quantitate the identified proteins. Such identification may be useful for a number of different things, for, in non-limiting examples, determining efficiency of purification, or identifying candidate binding molecules (by their absence or depletion).

As used herein, "immunoassay" refers to one of a number of techniques for the determination of the presence or quantity of a substance, especially a protein, through its properties as an antigen or antibody. The binding of antibodies to antigen is often followed by tracers, such as fluorescence or (radioactive) radioisotopes, to enable measurement of the substance. Immunological assays (immunoassays) have a wide range of applications in clinical and diagnostic testing. An example is solid- phase immunoassay in which a specific antibody is attached to a solid supporting medium, such as a PVC sheet. The sample is added and any test antigens will bind to the antibody. A second antibody, specific for a different site on the antigen, is added. This carries a radioactive or fluorescent label, enabling its concentration, and thus that of the test antigen, to be determined by comparison with known standards.

As used herein, "immunosubtractor" refers to affinity binding compositions, affinity columns, and apparati for affinity separations, non-limiting examples of which are described in US patent application publication number 20040115725 (the entirety of which is incorporated by reference). The term is also used herein to encompass the Multiple Affinity Removal System (MARS) of Agilent Technologies, Inc. (Palo Alto, CA) and its separation components, such as the Human Multiple Affinity Depletion (MAD) column (4.6 X 100 mm, capacity 30-40 μi of plasma). As used herein, "incorporate" means to become part of or be mixed with.

Incorporation need not mean that the properties of what is being incorporated are retained; neither need it mean that they are lost. However, preferably they are retained at least in part, such as the buffering capacity and vaporizability of a volatile buffer incorporated into a sample. As used herein, "interact" refers to binding, proteolyzing, modifying, regulating, altering, and the like. Generally it refers to direct interaction, but it may also refer to indirect interaction such as through a biochemical or genetic pathway.

As used herein, "in vitro binding assay" refers to an assay and/or a system for detecting and/or measuring, qualitatively and/or quantitatively, the binding between at least two substances, for example a protein, DNA, and/or RNA and another specific substance or complex, such a protein, DNA, RNA, cyclized peptide, or small molecule in vitro. The assay or system may be cell-based, such as in the yeast two hybrid and variants thereupon, or, for example, as in CAT or luciferase assays in cultured cells, and may be immunologically-based, such as with the use of immunoaffinity columns, ELISA assays, and the like. Assays utilizing a live animal or person are considered "in vivo". As used herein, "low solubility" refers to a species that one of ordinary skill in the art would describe as having a low solubility in water (property of being soluble, relative capability of being dissolved). For example, proteins with transmembrane domains are often considered to be of low solubility. Typically, such compounds are described in the Merck Index as "poorly soluble," "practically insoluble," "slightly soluble," "sparingly soluble," or using other similar terms. Generally, such compounds have a solubility of less than about 2 mg/mL at room temperature. Preferably, such compounds have a solubility of less than about 1 mg/mL.

As used herein, "mass spectrometer" refers to a gas phase ion spectrometer that measures a parameter which can be translated into mass-to-charge ratios of gas phase ions. Mass spectrometers generally include an inlet system, an ionization source, an ion optic assembly, a mass analyzer, and a detector. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. As used herein, "mass spectrometry" refers to a method comprising employing an ionization source to generate gas phase ions from an analyte presented on a sample presenting surface of a probe and detecting the gas phase ions with a mass spectrometer. Samples analyzed by mass spectrometry may undergo prior separation or treatments to enhance mass spectrometry results. As used herein, "method of screening" refers to a method suitable and typically used for testing a particular property or effect of a large number of compounds, including the identification and possible isolation of an individual compound or compounds based on a particular property such as binding or not binding to a target molecule. Typically, more than one compound is tested simultaneously (as in a 96-well microtiter plate) and, if desirable, portions of the procedure can be automated. "Method of screening" also refers to a method of determining a set of different properties or effects of one compound simultaneously. Screening may also be used to determine the properties for a complete set of compounds in a non-selective fashion, or may be used to select for a particular property or properties, such as might be desired to reduce the number of candidate compounds to be examined in later screening efforts or assays, Screening methods may be high-throughput and may be automated. As used herein, "multi-subuπit complex" refers to a complex comprising two or more biomolecules that are joined to the complex by binding interactions or part of the complex based on steric hindrance or containment. The biomolecules need not be all of a particular class of molecules, though protein complexes are preferred. The biomolecules may be the same biomolecule, in other words, the complex may be a multimer. Non-limiting examples of multi-subunit complexes include actin filaments, collagen, a cyclύvCDK-substrate complex, a cyclin-CDK-CDK inhibitor complex, RNA polymerase II complexed with DNA, aproteasome, and a mitochondria. For the purposes herein, "multi-subunit complex" may refer to the complex itself or some or all of its constituent parts.

As used herein, "non-binding proteins," "unbound proteins," or "non-binding molecules" are the proteins/molecules that are not retained by an affinity reagent under a given set of conditions for purifying a sample, for example, the proteins/molecules in the sample that are in the flow-through from an immunoaffinity column, such as a depleted plasma sample.

"Phenotype" refers to the form taken by some character (or group of characters) in an organism that is part of its observable structure, function, make-up, or behavior. Observation or determination of a phenotype may be at the social, organismal, organ, tissue, cellular, or molecular level. For example, a molecular phenotype may be exhibited by differences in protein complexes found in the cells of one organism versus another. The presence of a particular phenotype in an organism may be in part dependent on the genotype of the organism.

As used herein, "protein," "peptide," or "polypeptide" refers to any of numerous naturally occurring, sometimes extremely complex (such as an enzyme or antibody) substances that consist of a chain of two or more amino acid residues joined by peptide bonds. The chain may be linear, branched, circular, or combinations thereof. Intra-protein bonds also include disulfide bonds. Protein molecules contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur, and occasionally other elements (such as phosphorus or iron). Herein, "protein" is also considered to encompass fragments, variants and modifications (including, but not limited to, glycosylated, acylated, myristylated, and/or phosphoiylated residues) thereof, including the use of amino acid analogs, as well as non-protein acious compounds intrinsic to enzymatic function, such as co-factors, or guide templates (for example, the template RNA associated with proper telomerase function). In context, "protein" may be used to refer to a full-length (encompassing the whole of the coding sequence) or full-length post-translationally modified polypeptide as encoded by a particular nucleic acid sequence, and "peptide" may be used to refer to short amino acid sequences (roughly 2 to 50 amino acids) or non-fulHength polypeptide, but this should not be taken as limiting relative to the above definition,

For purposes of the invention a sample may be said to be "purified" or have undergone "purifying" when the amount of one or more contaminants or undesirable molecules is reduced, for example a blood plasma sample may be said to be

"purified" when it has been run through an immunodepletion column that removes albumin. Note that "purified" is a relative term with respect to a particular molecule or molecules. Thus, a plasma sample considered purified relative to the presence of albumin in the sample may not be considered purified relative to immunoglobulins. For purposes of the invention "purified" is considered relative to appropriate molecules that could be depleted using the affinity reagent or reagents employed in a particular embodiment of the invention. Purity can be measured by standard assays known in the art or described herein, examples of which include SDS-PAGE followed by Coomasie blue staining as well as chromatographic methods (e.g., size exclusion chromatography (SEC) on a HPLC system). A sample is considered pure if it is at least 90%, 95%, or 99% free of components other than the desired product.

"Reference sample" generally refers to a sample used as a control that is chosen to represent a normal or known state (e.g., a positive reference sample), or that is designated normal or known (e.g., positive reference sample) based on statistical evaluation. A reference sample may be used as a benchmark for assessment of a sample from which such benchmarks may be derived. Thus a reference sample may also be a sample chosen as representative of a particular condition or state, such as presence of a disease. Determination of appropriateness of use as a reference sample may be judged by one skilled in the art before or after measurement of the desired characteristics for which the sample will be used as a reference or as part of a population of reference samples, depending on the reasonableness to do so. For example, it may be reasonable for a group of patients to be designated as reference samples and "normal" for a mutant phenotype they do not display, and measurements of a panel of genes for gene expression may then be used as a reference range for normals relative to that phenotype, In another example, the reference level can be a level determined from a prior sample taken from the same subject. Or, for example, it may be reasonable to determine a protein's concentration in blood from a random sampling of the population (the reference sample thereby being a random sample) and using statistical methods delineate a normal range, or reference range. Or, a population of samples from untreated patients with melanoma and a population of patients with melanoma undergoing treatment might be useful in providing reference samples for comparison of the effects of a second therapy on protein expression levels. In some contexts, "reference sample" may simply refer to a sample of known quantity, of normal quantity, or readily detenninable quantity for comparison. Reference samples may be used to determine reference ranges and/or reference levels for characteristics of the samples. One skilled in the art may be able to determine an appropriate reference sample when one is desired.

As used herein, "sample" (or "biological sample") refers to any solid or fluid sample obtained from, excreted by, or secreted by any living organism, including single-celled micro-organisms (such as bacteria and yeasts) and multicellular organisms (such as plants and animals, for instance a vertebrate or a mammal, and in particular a healthy or apparently healthy human subject (e.g., a reference sample), a human patient affected by a condition or disease to be diagnosed or investigated), and those subjected to environmental or treatment conditions. A biological sample may be a biological fluid obtained from any location (such as whole blood, blood plasma, blood serum, urine, bile, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion), an exudate (such as fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (such as a normal joint or a joint affected by disease such as rheumatoid arthritis). Alternatively, a biological sample can be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (whether primary cells or cultured cells) or medium conditioned by any ceU, tissue, or organ. If desired, the biological sample is subjected to preliminary processing, including separation techniques. For example, cells or tissues can be extracted and subjected to subcellular fractionation for separate analysis of biomolecules in distinct subcellular fractions, e.g., proteins or drugs found in different parts of the cell. A sample maybe analyzed as subsets of the sample, e.g., bands from a gel. "Sample" may also be more broadly used to encompass recombinant, synthetic, and in vitro generated compounds or collections of compounds, and/or their combination with, or presence in, biological samples, for example, a protein complex produced and self-assembled in reticulocyte lysate by in vitro translation (IVT, e.g., Product # L4540, Flexi^® Rabbit Reticulocyte Lysate System, Promega, Madison, WI). Such samples may be useful as controls or in providing a desired set of experimental conditions, such as for a method of screening. As used herein, "semi-automated" refers to a method or procedure that is not completely automated; it still requires some human interaction after being initiated. For example, the plate-switching step, where the plate containing the samples is physically moved from one piece of equipment to another is done manually and other individual steps occur without manual manipulation. Thus, a semi-automated method differs from an automated method in that some of the steps outside of set-up are carried out manually.

A "solid support" is a material, essentially insoluble under the given solvent and temperature conditions, with which one or more affinity reagents is retained (attached, bound, disposed thereon) and/or made more easily separable from a sample the affinity reagents are brought into contact with. In a preferred embodiment, the solid support is covalently coupled to one or more affinity reagents capable of directly or indirectly binding a target molecule, such as a protein, When the target molecule is a protein, the affinity reagent preferably comprises an immunoaffinity reagent. The solid support is also preferably a particle such as a bead or sphere in the micron or submicron size range, referred to herein as "beads." Preferably beads are 200 microns or less, more preferably 150 microns or less, most preferably 100 microns or less. The solid support is preferably made of materials that may include one or more of the following: silica, polyacrylate, polyacrylamide, a metal, polystyrene, latex, nitrocellulose, exocellulose, dextran, agarose, sepharose, polypropylene, and nylon. Preferably, the solid support is able to be affected by a magnetic field. In such a case, the solid support may have a magnetite core. Other preferred forms of solid supports include filters, planar surfaces, and plate wells (such as those found in high- throughput plate formats, or used for ELISA). Preferably plates are relatively rigid or self-supporting to allow for easy handling during manufacturing and easy handling during use by the end user (a human or a robot). Preferably the plate may be made of polymeric (especially thermoplastic) materials, glass, metallic materials, ceramic materials, elastomeric materials, coated cellulosic materials, and combinations thereof such as epoxy impregnated glass mats. In a more preferable embodiment, the plate is formed of a polymeric material including but not limited to polyethylene, acrylic, polycarbonate, and styrene. The wells can be made by injection molding, drilling, punching, and/or any other method well known for forming holes in the material of selection- Such plates are well known and commercially available from a variety of sources in a variety of well numbers and designs. Most common are 96 and 384 well plates. Plates are typically 5 inches (127 mm) long and 3.4 inches (86.4 mm) wide. The plate thickness can vary but are generally 0.5 inches (12.7 mm) for a standard plate and 1.75 inches (44.45 mm) for a deep well plate. The well format will be determined by the end users needs, but it can have numerous configurations and the wells do not necessarily need to be all of the same shape or size. Especially with the smaller sized wells, the wells may have the same or different volumes. The wells may also have different shapes (e.g., round, rectangular, teardrop, square, polygonal, and other cross-sectional shapes or combinations of them). Virtually any shape that is required for the product may be provided. Typically, the wells arranged in uniformly spaced rows and columns for ease of use. Filters may be woven or non-woven, including but not limited to multilayer or composite filters. Not all layers of a multilayer filter need retain, bind, be attached to an affinity reagent. Filters can be chosen with respect to their properties in a way corresponding to the requirements of the respective sample and desired purification, so that the necessary purity class for the medium to be filtered is ensured. In a preferred way, the particle retention of the filters used is >60 micrometers, preferably >100 micrometers. Columns are also preferred solid supports, but are generally a secondary support retaining another form of support such as beads and filters. Solid supports may be used in any combination. For example a column may contain multiple compartments allowing flow through that contain different beads with different attached affinity reagents as well as filters with attached affinity reagents. The terms "specific binding" and "specific interaction" refer to binding and/or an interaction, even briefly, between one or more molecules, compounds, or complexes, wherein the binding and/or interaction is dependent upon the primary amino acid sequence (or other structural elements in a non-peptidic portion of a molecule), post-translational modifications to the amino acid sequence or its modifications, and/or their secondary structure or conformation. A molecule that exhibits specific binding toward another molecule may be said to be "specific for" the other molecule. Generally specific binding provides the ability for two molecular species concurrently present in a heterogeneous (non-homogeneous) sample to bind to one another preferentially over binding to other molecular species in the sample. In other words, "specificity" refers to the potential to bind one unique chemical structure more strongly than a number of similar alternatives. Typically, a specific binding interaction will discriminate over adventitious binding interactions in the reaction by at least two-fold, more typically more than 10- to 100-fold. When used to detect an analyte, specific binding is sufficiently discriminatory when determinative of the presence of the analyte in a heterogeneous (non-homogeneous) sample. Typically, the affinity or avidity of a specific binding reaction is least about 10^"* M, with specific binding reactions of greater specificity typically having affinity or avidity of at least 10^"6 M to at least about IO ¹² M. It may also refer to binding to self, or other molecules of the same protein, as in the forming of dimers and other multimers. Specific binding may also be used to connote a use in a discriminatory separation, diagnostic, or identification technique or a discriminatory property beyond simply recognizing the presence of the binding target in a sample; for example, an antibody may be considered discriminating of different members of a closely related protein family, for specific modified forms of a protein (e.g., a phosphorylated form vs. a non- phosphorylated form), or specific conformations of a protein. Specific binding may also be described as "recognition" or "recognizing" of a molecule by a binding molecule.

As used herein, 'Vacuum evaporation" refers to techniques for evaporation or removal of solvent from a sample. Common techniques for vacuum evaporation include rreeze-drying, centrifugal evaporation (such as use of a vacuum concentrator), rotary evaporation, vortex evaporation and lyophilization. As used herein, "volatile buffer" refers to a buffer having components with a vapor pressure (either a sum of vapor pressures of its components or/and the vapor pressure of the major component) that is grcter than or equal to 10 mm Hg at 25° C. In particular, with regard to the invention, the term is often used throughout to refer to a volatile binding buffer, which is a volatile buffer present when a sample is contacted with an affinity reagent and binding between the sample, or components thereof, and the affinity reagent is allowed or expected to occur. A variety of factors may influence the most appropriate volatile buffer to be used with a particular affinity reagent, such as the pH. Some examples of volatile buffer systems are: formic acid; pyridine / formic acid; trimethylamine / formic acid; pyridine / acetic acid; trimethylamine / acetic acid; trimethylamine / hydrochloric acid; ammonia / formic acid; ammonia / acetic acid; trimethylamine / carbonate; and ammonium bicarbonate. Preferable volatile buffer systems of the invention are those composed, at least in part, of ammonium ions.

Determination of viability of volatile buffers for use as binding buffers in immunoaffinity chromatography

The present inventors have developed a method for the use of volatile buffers, such as an ammonium bicarbonate buffer, m depletion experiments or sample preparation using affinity reagents, such as immunoaffinity depletion, that are useful for shortening a purification process, reducing the amount of reagents and materials, and improving recovery of the depleted sample proteins.

Any appropriate volatile buffer may be used. Preferred volatile buffer systems of the invention are those in part composed of ammonium ions — such as ammonium formate, ammonium acetate, or ammonium bicarbonate. pH, buffering range, ionic strength and capacity are factors in determining an appropriate buffer. The volatile buffer is used as a binding buffer in the methods of the invention, and may also be used as a buffer in subsequent steps of a separation such as being used as a wash or elution buffer. In particular embodiments, ammonium bicarbonate is present in a sample in concentrations of about 50 iuM, 100 mM, 150 mM, 200 mM, or 250 mM, or any concentration between about 50 mM and 250 mM. Volatile buffers are typically used at concentrations that safely allow their removal through solvent removal techniques, such as vacuum evaporation. Incorporation of volatile buffers into a sample may take place prior to or during a binding step, and may require the use of additional methods and techniques to adequately mix, resuspend, dissolve or incorporate the buffer into the sample (or the sample into the buffer), such as stirring, heating, agitating, and maybe performed by incorporating to intermediate concentrations before the final desired concentration is reached. Generally, the method includes the use of the volatile buffer as a binding buffer prior to purification of a sample. Thus, the method provides for purifying a sample including one or more proteins by incorporating a volatile buffer into the sample, contacting the sample with one or more affinity reagents, and separating the portion of the sample not specifically bound to the affinity reagent(s). Exemplary samples include one or more proteins, such as a complex mixture, a cell lysate, or a homogenized tissue. Other exemplary samples include blood samples (such as whole blood or blood serum), blood plasma samples, cerebrospinal fluid, saliva, urine, cell lysate, or cellular material.

Affinity reagents of the invention maybe any appropriate affinity reagents for a given procedure, for example anti-albumin antibodies for an immunodepletion of albumin from a blood sample. Combinations of reagents are contemplated for procedures that may use more than one affinity reagent to bind a particular target and/or to bind multiple targets. Affinity reagents may bind particular moieties such as structural determinants, discontinuous epitopes, linear epitopes, and post-translational modifications. Exemplary affinity reagents include immunoafϊϊnity reagents, particularly those used in immunodepletion. Affinity reagents of the invention can also include one or more solid supports, such as beads, filters, planar surfaces (e.g., a sheet of nitrocellulose, the inner wall of a column, the walls and/or bottom of a plate well, or coatings thereupon, etc. ~ planar surfaces may be curved planar surfaces), and other support forms, and combinations thereof.

The separation of the bound (generally preferably bound by one or more of the affinity reagents of the invention) from unbound sample may occur by any appropriate method without limitation, and may occur in steps. Generally a binding step will be used to allow binding to occur. After binding, additional steps such as washes or an elution step may take place, particularly if chromatography is the method of separation used. Preferred separation methods are chromatography and centrifugation. Most preferred is the use of an immunosubtractor, for example the MARS system of Agilent Technologies, Inc.

Preferably, the samples purified by the methods of the invention undergo a form of solvent removal. Solvent removal is accomplished by art-recognized methods, including one or more of the following: vacuum removal, evaporation, heating, centrifugal evaporation, rotary evaporation, vortex evaporation, lyophilization, dialysis, liquid-liquid separation, solid-liquid separation, and/or precipitation of the material followed by separation of the precipitate and the solvent. In one embodiment, a solvent is removed by vacuum evaporation in combination with freeze-drying, In large-scale preparations, solvent removal includes a standard continuous evaporation process such as described by Fosslien and Musil ((1970) J Lipid Res. 11: 605 - 609). In another embodiment, solvent is removed after precipitation of solute proteins. Such precipitation processes are well known in the art, and may include but are not limited to one or more of the following: addition of a precipitant, such as a salt or a further solvent, change in temperature, and/or change in pH. Following precipitation, the liquid phase may be decanted, filtered, evaporated or removed by means as are known to those skilled in the art.

In one embodiment of the invention, the method is semi-automated to aid in increasing reproducibility in the methods of the invention, reducing labor, reducing time, and reducing cost. In another embodiment the methods of the invention are fully automated.

In another embodiment, samples prepared by the methods of the invention are subjected to analysis by mass spectrometry. Analysis by mass spectrometry may be useful in, for example, identifying and/or quantitating proteins in a sample and aiding in determining the composition of a sample. For example, blood plasma samples purified by the methods of the invention could be used for analysis by mass spectrometry for the identification of peptide or protein biomarkers associated with a disease, symptom, or phenotype. Additional separations of biomolecules may be performed subsequent or prior to performing the methods of the invention and prior to analysis by mass spectrometry. A wide variety of techniques for separating any of the aforementioned biomolecules are well known to those skilled in the art (see, for example, Laemmli (1970) Nature 227: 680 - 685; Washbuπi et al. (2001) Nat. Biotechnol. 19: 242 - 247; Schaggeret et al. (1991) Anal. Biochem. 199: 223 - 231) and may be employed according to the present invention.

In one application, the methods of the invention are used to study complex mixtures of proteins. By way of example, mixtures of proteins are separated on the basis of isoelectric point (e.g., by chromatofocusing or isoelectric focusing), of electrophoretic mobility (e.g., by non-denaturing electrophoresis or by electrophoresis in the presence of a denaturing agent such as urea or sodium dodecyl sulfate (SDS), with or without prior exposure to a reducing agent such as 2-mercaptoethanol or dithiothreitol), by chromatography, including LC, FPLC, and HPLC, on any suitable matrix (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, or affinity chromatography, for instance with an immobilized antibody or lectin or immunoglobins immobilized on magnetic beads), or by centrifugation (e.g., isopycnic centrifugation or velocity centrifugation). In some cases, two different peptides may be detected as having similar masses within the resolution of a mass spectrometer, rendering determination of abundances for those two peptides difficult. Separating the peptides before analysis by mass spectrometry allows for the resolution of the abundances of two peptides with the same mass. Although many spectra for the fractions of the separation may then be obtained, these spectra typically have a reduced number of ion peaks from the peptides, simplifying the analysis of a given spectrum.

In one embodiment, a mixture of proteins is separated by ID gel electrophoresis according to methods known in the art. The lane on the gel containing the separated proteins is excised from the gel and divided into fractions. The proteins are then digested enzymatically. The peptides produced in each fraction are then analyzed by mass spectrometry. In another embodiment, peptides are separated by 2D gel electrophoresis according to methods known in the art. The proteins are then digested enzymatically, and the digested peptides produced in each fraction are then excised and analyzed by mass spectrometry. In still another embodiment peptides are separated by liquid chromatography (LC), e.g., multidimensional LC. LC fractions may be collected and analyzed or the effluent may be coupled directly into a mass spectrometer for real-time analysis. LC may also be used to separate further the fractions obtained by gel electrophoresis. Recording the retention time (RT) of a peptide in LC enables the identification of that peptide in multiple fractions. This identification is typically useful for obtaining an accurate abundance. In any of the above embodiments, a given peptide may be present in more than one fraction depending on how the fractions were obtained. Exemplary methods for analyzing biomolecules using mass spectrometry techniques are well known in the art (see, for example, Godovac-Zimmermann et al. (2001) Mass Spectrom. Rev. 20: 1 - 57; Gygi et al. (2000) Proc. Natl. Acad. Sri. USA 97: 9390 - 9395).

In applications involving peptides, the peptides are ionized, e.g., by electrospray ionization, before entering the mass spectrometer, and different types of mass spectra, if desired, are then obtained. The exact type of mass spectrometer is not critical to the methods disclosed herein. For example, in a survey scan, mass spectra of the charged peptides in a sample are recorded- Furthermore, the amino acid sequences of one or more peptides may be determined by a suitable mass spectrometry technique, such as matrix-assisted laser desorptioπ/ionization combined with time-of-flight mass analysis (MALDI-TOF MS), electrospray ionization mass spectrometry (ESI MS), or tandem mass spectrometry (MS/MS). In a MS/MS scan, specific ϊons detected in the survey scan are selected to enter a collision chamber. The ability to define the ions for MS/MS allows data to be acquired for specific precursors, while potentially excluding other precursors. The ions may be defined by a predetermined list or by a query. Lists may be inclusion lists (i.e., ions on the list are subjected to MS/MS) or exclusion (i.e., ions on the list are not subjected to MS/MS). The series of fragments that is generated in the collision chamber is then analyzed again by mass spectrometry, and the resulting spectrum is recorded and may be used to identify the amino acid sequence of the particular peptide. This sequence, together with other information such as the peptide mass, may then be used, e.g., to identify a protein. The ions subjected to MS/MS cycles may be user defined or determined automatically by the spectrometer.

In another embodiment, samples prepared by the methods of the invention are subjected to analysis by fluorescence. Analysis by fluorescence may be useful in, for example, identifying and/or quantitating proteins in a sample and aiding in determining the composition of a sample. For analysis by fluorescence, the proteins in the sample can be fluorescently labeled before or after the addition of the volatile buffer using standard methods known in the art. If the proteins are labeled after the addition of the volatile buffer, they can be labeled before or after the immuπodepletion step. Samples can then be subjected to analysis by fluorescence imaging techniques using standard methods known in the art.

The invention also provides methods of screening, including methods for identifying the proteins in a sample that are not specifically bound by an affinity reagent (by this is meant not that an affinity reagent specific for the protein does not exist, but that the focus of the screening method is on the protein(s) not removed from a sample by an affinity reagent in the separation step of the method, for example, if an affinity reagent to protein X is used and X, Y, and Z form a protein complex in some patients but not others, V and Z may be identified as proteins "not specifically bound by an affinity reagent," in samples where the XYZ complex does not stably form). This method includes incorporating a volatile buffer into the sample, contacting the sample with one or more affinity reagents, separating the portion of the sample not specifically bound to the affinity reagent(s), and identifying one or more of the non- binding proteins. Thus, surveying a population of samples may be perfumed, and as such is useful for identifying samples to focus on based on particular characteristics, or particular proteins to focus on based on the samples they were present in, etc. This method may be particularly advantageous in samples for which sample loss in processing is a critical factor, such as when the sample amount is very limited, or when cost of generating the samples is optimized by requiring a minimal amount of sample. Identification of samples which have altered activity in a functional assay, for example, samples that exhibit increased kinase activity in a kinase assay, for use in the method of screening may be used to focus on identifying the protein compositions of samples associated with the change in activity. This allows for the identification of key regulatory interactions impacting function or the identification of a protein composition useful as a desired endpoint in screening for compounds that alter the particular activity. For example, if the protein complex XYZ generally present in cells is observed in a functional assay to have increased activity when Z is no longer part of the complex, it may be possible to screen for compounds that increase the XYZ activity by screening for compounds that after separation using a protein X binding affinity reagent yield a sample that does contain Y but does not contain Z by immunoassay instead of a potentially more costly or labor intensive activity assay. Or, by screening to identify compounds that alter activity but do not impact protein composition, it may be possible to screen for compounds that may have potentially fewer toxic or undesired side effects.

The methods of screening may also be useful, for example, in performing a "virtual subtraction," i.e., for direct comparison of non-binding proteins in a proteome or sample across a variety of conditions to determine which proteins may be differentially expressed or bound under the various conditions. Thus, in one embodiment, the invention includes a method for identifying differences in the protein composition of one or more samples, and the method includes incorporating a volatile buffer into the sample, contacting the sample with one or more affinity reagents, separating the portion of the sample not specifically bound to the affinity reagent(s), identifying one or more of the proteins present in the portion of the sample not specifically bound to the affinity reagent(s), and determining the relative composition of a sample to one or more other samples. Determining the relative composition of a sample as compared to one or more other samples, such as a reference sample or population of reference samples, can include determining the expression levels of one or more peptides in the sample and comparing the expression levels of the one or more peptides from the sample to the expression levels in the reference. Differences in the relative abundance of the peptides from the sample to the reference can be used to identify differentially expressed peptides. For example, using a phosphate-binding affinity reagent, cell lysate samples may be compared after different kinase pathways have been activated and to a reference sample for differences in non-binding proteins, thereby identifying non-phosphorylated proteins. Multi-subunit complex integrity can also be screened in this manner to determine if particular parts, or all of a complex or complexes, may be present. For example, a complex that includes a dimer of protein T plus proteins U, V, and W could be screened under various conditions to determine through quantitation of abundance when T is a monomer in the complex, when U ajid V are simply not part of the complex, when the complex is totally disrupted, and/or when such states correlate with a screen for functional activity (e.g., kinase activity is only present when protein W is glycosylated and protein V is absent from the complex). Such screens maybe useful particularly in screening for small molecules, for example, those that disrupt a function but maintain complex integrity. In another example, a multi-subunit complex's integrity, or partial or complete disruption of the integrity, by members of a library of compounds may be tested in cell lysate or cellular material. This may be useful, for example, in determining which compounds bind to a target molecule without disrupting the whole complex, and may involve numerous samples, as well as numerous affinity reagents specific for one or more components of the complex. In addition, such screening may be useful in determining the stability of a complex in certain solutions. Order of multi-subunit assembly could similarly be addressed by adding individual components in different orders.

The determination of relative composition of a sample may be qualitative, quantitative, or both. For example, for samples analyzed by mass spectrometry LC- MS scans may be compared as might quantities of individual peaks of the scans and the peptides for which amino acid sequence was obtained by tandem mass spectrometry. Comparisons may be between any samples or within or between groups of samples. Comparisons may be, for example, between samples of different genotypes but the same genetic background, samples of different phenotypes, such as those exhibiting different functional activities, between normal samples and tumor samples, between samples from a patient with a disease and a healthy patient, between a patient with a disease and the same or another patient undergoing treatment or across particular time points or at particular disease stages, or between any sample and a control or reference sample. The comparison may between all the components of the sample, only the identified components of a sample, or specific components of the sample, For example, molecules of particular m/z and retention time maybe compared, or just the identified transmembrane domain containing peptides. The determined composition may be used in future comparisons as a "profile," and/or a subset there of may be identified for use as a biomarker or surrogate endpoint. The methods of the invention can also be used to screen for conditions which alter the protein composition of one or more samples as compared to one or more other samples, wherein said proteins are not specifically bound by an affinity reagent. Conditions are the parameters set for variables of an experiment, such as treatment, non-treatment, the addition to or presence of one or more compounds in the sample, temperature, time of contact, mixing, genotype, genetic background, air pressure, salt concentration, buffers used, etc, but may also refer to the equivalent variables present in a population. For example, samples may come from cells of different genotypes or exhibiting different phenotypes. The method includes: incorporating a volatile buffer into the sample, contacting the sample with one or more affinity reagents, separating the portion of the sample not specifically bound to the affinity reagent(s), identifying one or more of the proteins present in the portion of the sample not specifically bound to the affinity reagent(s), determining the relative composition of the sample as compared to one or more other samples, and determining the conditions under which the protein composition of the sample is altered.

Libraries (e.g., protein libraries) for the screening methods of the invention can be obtained using any of the numerous suitable approaches in combinatorial library methods known in the art, including; biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145 -167; US Patent Nos. 5,738,996 and 5,807,683).

Examples of methods for the synthesis of molecular libraries that may be used in the invention's methods of screening can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909 - 6913; Erb et al. (1994) Proc. Natl. Acad. Sci. US A 91 : 11422 - 11426; Zuckermann et al. ( 1994) J . Med. Chem. 37: 2678 - 2685; Cho et al (1993) Science 261: 1303 - 1305; CareU et al. (1994) Angew, Chem. Int. Ed. Engl. 33: 2059 - 2061 ; Carell et al. (1994) Angew. Chem. Int. Ed, Engl. 33: 2061 - 2064; and Gallop et al. (1994) J, Med. Chem. 37: 1233 - 1251. Libraries of compounds may be presented in the methods of screening, e.g., presented in solution (e.g., Houghten (1992) Biotechniques 13: 412 - 421), or on beads (Lam (1991) Nature 354: 82 - 84), chips (Fodor (1993) Nature 364: 555 - 556), bacteria (US Patent No. 5,223,409), spores (US Patent Nos. 5,571 ,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865 - 1869) or phage (Scott and Smith (1990) Science 249: 386 - 390; Devlin (1990) Science 249: 404 - 406; CwMa et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378 - 6382; andFelici (1991) J. MoI. Biol. 222: 301 - 310).

The invention also provides a kit that includes a volatile buffer and instructions for the use of the volatile buffer as a binding buffer. The kit can be used for any of the immunoafϊϊnity methods described herein.

EXAMPLES Chemicals and reagents

The following is a list of chemicals and reagents used in the Examples described herein. Buffers A (binding) and B (elution) for the Multiple Affinity Removal System (MARS) were obtained from Agilent Technologies, Inc. (Palo Alto, CA, USA)

Ammonium bicarbonate and hydrochloric acid (HCl) were bought from EMD Chemicals, Inc. (Gibbstown, NJ, USA)

Gels (4-12% Bis-Tris), 10 x MES running buffer, Sypro Ruby stain and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample preparation reagents were bought from Invitrogen Canada, Inc. (Burlington, Canada).

Human Haptoglobin, C-3 and C-4 complement proteins, human α-1 -antitrypsin and the following antibodies: Goat Anti-C3, Mouse anti-α-l -antitrypsin and Goat anti- rransferrin were bought from VWR CanLab (Mississauga, ON, Canada). Human Apo-transferrin, Human Serum Albumin and Tris-base were bought from Sig-na-Aldrieh Canada, Ltd. (Oakville, ON, Canada).

Other reagents were supplied by the following manufacturers: Water, HPLC grade, by Fisher Scientific (Montreal, Quebec, Canada); Human IgG by Bethyl Laboratories, (Montgomery, TX, USA);

Sheep polyclonal anti-alburnin by US Biologicals (Swampscott, MA, USA);

Goat Anti-IgG and Goat Anti-Kappa light chain, both anti-Human by Southern Biotechnology Associates (Birmingham, AL, USA); Sheep polyclonal anti-haptoglobin antibodies by Abeam (Cambridge, MA, USA);

HRP-conjugated AffiniPure Rabbit anti-sheep, anti-goat and Goat anti-mouse by Jackson Immunoresearch, Inc. (West Grove, PA, USA); and,

Human plasma, EDTA-treated, was bought from Bioreclamation, (NY, USA).

A standard solution of BSA (2 mg/ml), and reagents A and B, for a Bicinchoninic Acid base protein assay (BCA) were obtained from Pierce, (Rockford, IL, USA).

Unless otherwise specified, all solvents and chemical reagents were of analytical grade.

Materials and Equipment The following is a list of methods and equipment used in the Examples described herein.

A Human Multiple Affinity Depletion (MAD) column (4.6 X 100 mm, capacity 30-40 μl of plasma) for MARS was obtained from Agilent Technologies, Inc. (Palo Alto, CA, USA). Chromatograph "AKTA explorer" was bought from Amersham Biosciences (Uppsala, Sweden).

Immulon 4HBX 96-well plates were bought from Dynex (Chantilly, Virginia, USA).

PowerWave plate reader was bought from BioTek (Wobum, MA, USA). Dialysis cassettes were purchased from Pierce (Rockford, IL, USA). Example 1. Comparison of the depletion efficiency under volatile and nonvolatile conditions

Forty μl of a thawed human plasma aliquot were mixed with either 160 /xl of 150 mM ammonium bicarbonate (for the depletion in volatile buffer) or 160 ul of Agilent Buffer A (for the depletion in non-volatile buffer). Two samples were prepared for the depletion under volatile conditions and one sample for the depletion under non-volatile conditions. Diluted samples were briefly vortexed and then spun for 1 minute at 10000 x g in a microcentrifuge (Mioromax RF, Fisher Scientific, Montreal, Canada). One hundred microliters of the supernatant were used for the depletion. All samples were prepared individually in identical time frames before an injection.

Samples prepared in ammonium bicarbonate buffer were loaded onto the pre- equilibrated column in the stream of 150 mM ammonium bicarbonate buffer at a flow rate 0.5 ml ^• min^"1. Unbound proteins (depleted plasma) were collected. At the 7^tb minute of the run, the gradient was switched to 100% Agilent buffer A and continued for 4.5 minutes at 1 ml ^■ min^'1. Bound proteins were then eluted with Agilent buffer B for 4.5 minutes at 1 ml • min^"1, and the column was washed with Agilent buffer A for 4.5 minutes at 1 ml ^• min^"' . The column was equilibrated in 150 mM ammonium bicarbonate for 5.5 minutes at 1 ml ^• min^'1. The sample prepared in Agilent buffer A was loaded onto the pre-equilibrated column in the stream of Agilent buffer A at flow rate 0.5 ml ^• min^"1. Unbound proteins (depleted plasma) were collected. At the 1 I^th minute of the run, the gradient was switched to 100 % of Agilent buffer B and the flow rate increased to 1 ml ^• min^"1. After 4.5 minutes the gradient was switched to Agilent buffer A, and the column was equilibrated for 10.5 minutes at a flow 1 ml ^• min^"1.

One of the two samples prepared using volatile buffer and the sample prepared using non-volatile buffer were each transferred into separate 3 ml dialysis cassettes using a sample volume of 2.25 ml each. They were then dialyzed in the same bucket against 4 liters of water for 16 hours at 4° C with stirring. After the dialysis, the content of cassettes was transferred into tubes and freeze-dried. The remaining sample prepared using volatile buffer was directly freeze-dried. For freeze-drying, tubes containing the plasma samples were incubated in a freezer at -85°C for 45 minutes and then dried under vacuum for 16 hours. Dried samples were reconstituted in 400 μi of 100 mM ammonium bicarbonate, and freeze- drying was repeated. Dried samples were diluted in 10 mM Tris-HCl, pH 7,8 prior to instrumental analysis. Unless otherwise stated, after freeze-drying, all selected samples (including raw plasma samples) were analyzed by BCA, SDS-PAGE, and ELISA.

BCA analysis was executed according to manufacturer's directions. Briefly, 10 μl of duplicated serial dilutions of Bovine Serum Albumin (BSA) or analyzed samples were mixed with 200 microliters of BCA reagents in wells of 96-well plates. After incubation for 60 minutes at 37°C, plates were read at 562 nm in PowerWave microplate reader.

For SDS-PAGE analysis, 10 μg of protein (as per BCA assay) from each analyzed sample, including raw plasma, was prepared accordingly to manufacturer's directions (with reduction and boiling) and loaded onto 4-12% Bis-Tris gels. Gels were run in MES buffer for 30 minutes at 185 volts and stained overnight with Sypro Ruby.

ELISA analyses were performed for each analyzed sample in duplicates followed standard protocols. Briefly, aliquots from serial dilutions of analyzed samples were transferred into wells of ELISA plates for binding. After incubation, washing and blocking, wells were incubated with primary, then with secondary antibodies. Color development was monitored at 450 nm after a 4 minute incubation. Standard curves were constructed from readings of 8 duplicated serial dilutions in the range from 12.5 to 0-098 nanograms of protein per well. Commercial proteins were used to build standard curves for each protein of interest.

Based on the data obtained, plasma samples appear to be depleted with similar efficiency in the standard, non-volatile buffer conditions as in the volatile buffer. For example, in the ELISA results shown in Table 1, below, the percentage of protein depletion was equal to or greater than 99.5 % for albumin, alpha- 1 -antitrypsin, transferrin and IgG in dialyzed samples independent of depletion conditions. SDS- PAGE analysis (data not shown) also supports this observation. Table 1. Comparison of the depletion efficiency in volatile and non-volatile conditions.

*** Calculated as ((amount in raw plasma-amount in depleted plasma) x 100%)) / (amount in raw plasma).

**** Equivalent to 20 μl of raw plasma.

Table 1 shows BCA and enzyme linked immunosorbant assay (ELISA) measurements of total protein, quantities of individual proteins that are targeted for depletion by the column (albumin, IgO, alpha- 1 -antitrypsin, IgA (not directly quantitated), transferrin, and haptoglobin are also shown). Kappa light chains are also quantitated as a broader measure of Ig proteins. The measurements shown are for samples prepared under volatile conditions, both with and without dialysis before freeze-drying, and non-volatile conditions with dialysis before freeze-dryiπg. Measurements have been converted to % depletion where indicated.

Levels of C3 and C4 complement proteins were measured by ELISA in order to determine the loss of non-targeted proteins during depletion (non-specific loss). These two proteins were chosen because they are known common components of blood plasma for which ELISA adapted antibodies are commercially available. The sample depleted under volatile conditions and processed without dialysis has higher protein content (228.8 μg vs.l 14,1 μg) and lower non-specific losses than the dialyzed sample (37.7 % and 49 % depletion of C3 and C4 complement proteins, respectively, vs. 60 % and 76.7 % for the dialyzed sample) as shown in Table 1. Overall, the depletion with volatile conditions and without dialysis shows the smallest, though still dramatic, loss of C3 and C4 complement proteins.

High non-specific losses seem to be associated with only a few early runs in the column's history. All later runs (reproducibility experiments were executed on the same column using the same batch of frozen plasma samples) show much higher protein recovery (C3 complement, Table 3) than in runs executed earlier (Table 1).

Such behavior of previously unused chromatography columns is not abnormal.

Generally, instructions provided by the column manufacturers propose a few "polishing" runs with real samples to block sites of irreversible binding. On this column, more than 5 injections of plasma samples were required to limit non-specific losses to a minimal and reproducible level.

The use of volatile buffer neither affects the retention time or the shape of the peak of depleted plasma proteins (unbound proteins, Figure 1). However, it increases the chromatography time by approximately 5 minutes as compared to the original methodology of Agilent Technologies. The increase in run time was caused by the incompatibility of ammonium bicarbonate buffer with Agilent Technologies' buffer B (mixing of both causes excessive bubble production). Therefore, a plug of Agilent buffer A was always used between ammonium bicarbonate buffer and Agilent buffer B.

Example 2 Reproducibility of the depletion under volatile conditions

The Multiple Affinity Removal System (MARS) from Agilent Technologies was used in this study. This system enables removal of albumin, IgG, alpha- 1- antitrypsin, IgA, transferrin and haptoglobin from human blood plasma in a single step, through use of antibodies against these proteins covalently immobilized to a matrix that is packed into regular HPLC hardware. Based on SDS-PAGE analysis, Agilent Technologies claims that a single MARS column can be used for multiple (up to 200) injections with, high degree of mn to run reproducibility. The reproducibility (from run to run and from day to day) of the depletion process under volatile conditions is determined by a few factors. These include: column stability in volatile buffer, stability of the volatile buffer during the day of analysis, reproducibility of the buffer preparation from day to day, reproducibility of sample preparation and sample stability. For this study, samples were prepared simultaneously, and processed (freeze-dried) immediately after depletion.

A pool of human plasma samples was spun at 10000 x g for 1 minute, and the supernatant was distributed into 10 aliquots of 220 μl of plasma and frozen at -85⁰C. On the day of analysis, one frozen aliquot of plasma was thawed for 3 minutes in water at ambient temperature and diluted with 1100 μl of ice-cold 150 mM ammonium bicarbonate. The mix was briefly vortexed and distributed into 11 tubes of 120 μl each. Samples were stored on ice and injected over a period of approximately 5 hours at time intervals of approximately 30 minutes. Each aliquot was spun at 10000 x g for 1 minute immediately before injection and 110 μl of the supernatant was loaded into the 100 μl injection loop. Samples were manually loaded onto a pre-equilibrated column in a stream of

150 mM ammonium bicarbonate buffer at a flow rate of 0.5 ml • min^"1. Unbound proteins (depleted plasma) were collected. At the 7^t minute of the run, the gradient was switched to 100% Agilent buffer A and continued for 4.5 minutes at a flow rate of 1 ml ^■ min^"1. Then, bound proteins were eluted with a 100% Agilent buffer B gradient for 6 minutes at 1 ml ^• min^"1 and the column was washed with Agilent buffer A for 3 minutes at 1 ml ^■ min^"1. The column was equilibrated in 150 mM ammonium bicarbonate for 6 minutes at 1 ml/min.

One hundred runs (the 20^th to the 120^th in the column's history) were executed in increments of 10 injections per day. The first and each 20^lh run were analyzed. Therefore, each analyzed run (except for run # I) was the last (10^th) injection executed per day. Unbound (depleted plasma) and bound fractions were collected and frozen immediately. Frozen aliquots were further subjected to two cycles of freeze-drying: tubes containing plasma samples were incubated in a freezer at - 85°C for 45 minutes and then dried under vacuum for 16 hours, subsequently the dried samples were reconstituted in 400 μl of 100 mM ammonium bicarbonate and freeze-drying was repeated.

Dried samples were diluted in 10 mM Tris-HCl, pH 7.8 prior to instrumental analysis. Unless otherwise stated, after freeze-drying, all selected samples (including raw plasma samples) were analyzed by BCA, SDS-PAGE, and ELISA per the methods described in Example 1. The run to run and day to day reproducibility of the depletion of 100 injections of plasma was monitored by UV at 280 πm (Table 2, below). Standard deviations (SD) and coefficients of variation (CV) were calculated for each day and for the whole experiment (10 days) using peak heights of unbound and bound proteins. The day to day reproducibility of the depletion was also monitored by BCA assay and ELISA (Table 3) of the first and every subsequent 20^th run.

Table 2. Reproducibility of the depletion in the volatile buffer system by peak heights at 280 nm.

* Calculated based on days average values.

Table 2 shows the run to run and day to day reproducibility of the depletion of 100 injections of plasma as monitored by UV at 280 nm. Standard deviations (SD) and coefficients of variation (CV) were calculated for each day and for the whole experiment ( 10 days) using peak heights of unbound and bound proteins.

The peak heights of unbound fractions in the (JV analysis were more reproducible from run to run then from day to day. As can be seen in Table 2, the lowest reproducibility of peak heights (SD = 14.6, CV =^■ 1.6 %) comparing run to run, was observed on the 9^th day and the highest (SD = 6, CV = 0.6 %) on the second day of the study. A trend of decreasing peak heights of unbound fractions was observed from the 5* to the 9^th day of the experiment (Table 2). This trend was also observed for ratios of unbound vs. bound peak heights. No trends of peak heights were observed from run to run within any particular day.

The standard deviation for the whole study calculated from day averages of peak heights was higher (SD - 23.5, CV = 2.5 %) than for any particular day. This UV analysis observation was further supported by the BCA results (Table 3). The amount of protein in depleted plasma samples in runs from different days varies within approximately the same CV (2.7 %) limits.

Table 3. Reproducibility of the depletion in the volatile buffer system by ELISA and BCA.

Table 3 shows the day-to-day reproducibility of the depletion as monitored by BCA assay and ELISA (see Table 1 for information regarding the particular proteins quantitated) of the first and every subsequent 20^lh run for the same experiment as Table 2. Percent recovery and coefficients of variation (CV) are provided. Quantities of an individual protein (C3 complement) not targeted for depletion are also shown.

The data indicate that a MARS column shows higher reproducibility and lower non-specific losses than disposable IA cartridges (Ho et al. (2004) J. Assoc. Labb. Autom. P: 23S). SDS-PAGE analysis of unbound fractions revealed reproducible protein profiles from day to day. Protein patterns were similar to those of previously published results (Liao et al (2004) Arthritis Rheum. 50: 3792).

BCA and ELISA analyses of protein content in the samples of depleted plasma also show reproducibility from day to day in depletion efficiency and protein recovery (Table 3). Thus, the average amount of protein in depleted plasma samples obtained on different days was 284.2 μg with a CV of 2.7 %. The recovery of C3 complement was 100 % on average, with a CV of 10.3 % (Table 3). An approximately 4-fold, and relatively reproducible (CV = 10%), enrichment of C3 complement protein (Table 4) was observed as a consequence of removal of over 75% of the total protein content of the raw plasma (Table 3).

Table 4, Relative abundance of proteins before and after depletion.

* Calculated as (amount of a protein by ELISA / total protein content in the sample) x 100 %.

Table 4 shows the percent relative abundance in the sample before and after depletion for the proteins quantitated in Table 3. Standard deviations (SD) are provided.

A positive trend in kappa light chain and total IgG content in depleted samples was observed in the second half of the runs (Table 3). Thus, the amount of IgG and K- light chains increased from 0.47 and 1.5 μg per depleted sample, respectively, on the first day to 0.9 and 4.5 μg per depleted sample, respectively, on the 10^th day. The relative abundance of these two proteins increased two-fold during the ten day period and achieved 1.5 % and 0.3 % of total protein for κ-light chains and IgG, respectively. It could not be determined at the time of the study whether this was related to deteriorating antibodies from the column or to the decrease in IgG depletion efficiency.

Example 3 Use of Volatile Binding Buffers with Other Systems

The depletion in ammonium bicarbonate buffer can be implemented with other affinity matrices. The inventors have successfully applied it for the depletion of blood plasma proteins with protein-G, -A and -L resins.

Example 4 Semi-automation of Depletion Using Volatile Binding Buffers

Given that the depletion in a volatile buffer is reproducible from run to run (Example 2), a sequential, unattended depletion of multiple samples may be executed. The depletion process for larger samples or sets of samples may be executed in batches of runs over an extended period. Blood plasma contains a pool of very active proteases able to degrade a significant proportion of the total pool of plasma proteins within one hour. For an unattended depletion process executed in a sequential manner, samples can be prepared, depleted one after another, and processed after depletion simultaneously. With the samples in a liquid format for a time required to process all of them, sample stability needs to be maximized. A procedure which diminishes intrinsic protease activity in plasma samples for a period of 16 hours and does not interfere with an enzymatic digestion step in later stages of sample processing for mass spectrometry was used, allowing execution of unattended multiple sequential depletion runs.

Example 5 Use of Volatile Binding Buffers in Sample Preparation for Mass Spectrometry

Mass spectrometrtc analysis was used to confirm the high degree of the depletion reproducibility (Example 2). Other Embodiments

It will be clear that the invention may be practiced other than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the claims. Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. All references (e.g., patents, patent applications, including U.S. provisional patent application number 60/682,746, journal articles, abstracts, laboratory manuals, books, or other disclosures) mentioned herein are hereby incorporated by reference.

Claims

Claims

1. A method for purifying a sample comprising one or more proteins, said method comprising: a) incorporating a volatile buffer into the sample; b) contacting the sample with one or more affinity reagents; and c) separating the portion of the sample not specifically bound to the one or more affinity reagents, thereby purifying the sample.

2. The method of claim 1, further comprising the step of: d) removing solvent from the purified sample of step (c).

3. The method of claim 2, wherein said solvent is removed by vacuum evaporation.

4. The method of claim 3, wherein the purified and vacuum evaporated sample is further subjected to analysts by mass spectrometry.

5. The method of claim 1 , wherein the volatiIe buffer comprises ammonium ions,

6. The method of claim S, wherein the volatile buffer comprises ammonium formate.

7. The method of claim 6, wherein the volatile buffer is ammonium formate.

8. The method of claim 5, wherein the volatile buffer comprises ammonium acetate.

9. The method of claim 8, wherein the volatile buffer is ammonium acetate.

10. The method of claim 5, wherein the volatile buffer comprises ammonium bicarbonate,

11. The method of claim 10, wherein the volatile buffer is ammonium bicarbonate.

12. The method of claim 10, wherein the ammonium bicarbonate is present in a concentration of about 50 mM to 250 mM.

13. The method of claim 12, wherein the ammonium bicarbonate is present in a concentration of about 250 mM.

14. The method of claim 12, wherein the ammonium bicarbonate is present in a concentration of about 200 mM.

15. The method of claim 12, wherein the ammonium bicarbonate is present in a concentration of about 150 mM.

16. The method of claim 12, wherein the ammonium bicarbonate is present in a concentration of about 100 mM,

17. The method of claim 12, wherein the ammonium bicarbonate is present in a concentration of about 50 mM.

18. The method of claim 1 , wherein the one or more proteins in the sample have low solubility.

19. The method of claim 1, wherein the total protein concentration in the sample is less than 0.1 microgram / microliter.

20. The method of claim 1, wherein at least one affinity reagent is attached to at least one form of solid support

21. The method of claim 20, wherein said at least one form of solid support is a bead, a filter, a plasma surface, or a well of a multi-well plate.

22. The method of claim 1, wherein the at least one affinity reagent is an immunoaffinity reagent.

23. The method of claim 1 , wherein the separation method is centrifugatioH)

24. The method of claim 1 , wherein the method for purifying said sample is semi-automated.

25. The method of claim 1 , wherein the method for purifying said sample is automated.

26. The method of claim 1, wherein the sample is cellular material or a bodily fluid.

27. The method of claim 26, wherein the bodily fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, and cerebrospinal fluid.

28. The method of claim 26, wherein the bodily fluid is cerebrospinal fluid.

29. The method of claim 26, wherein the bodily fluid is saliva.

30. The method of claim 1 , wherein the sample is cell lysate.

31. The method of claim 1 , further comprising identifying differentially expressed peptides in said sample as compared to a reference, said method comprising: d) measuring the expression levels of one or more peptides in said sample; and e) comparing the expression levels of the one or more peptides from said sample to the expression levels of the one or more peptides from said reference; wherein differentially expressed peptides are identified based on the relative abundance of the one or more peptides in the sample as compared to the abundance of the one or more peptides in the reference.

32. The method of claim 31 , wherein the sample is identified as having altered activity in a functional assay as compared to the reference.

33. The method of claim 31 , wherein the reference is a normal control sample.

34. The method of claim 31, wherein said method is used to screen for conditions that alter the expression levels of one or more peptides in the sample as compared to said reference.

35. A method for identifying the proteins in a sample, said method comprising: a) incorporating a volatile buffer into the sample; b) contacting the sample with one or more affinity reagents; c) separating the portion of the sample not specifically bound to the one or more affinity reagent(s); and d) identifying one or more of the proteins present in the portion of the sample not specifically bound to the one or more affinity reagent(s).

36. The method of claim 35, wherein said samples comprise samples identified as having altered activity in a functional assay, thereby identifying changes in protein composition of the sample potentially associated with the change in activity.

37. The method of claim 35, wherein the one or more proteins identified in step (d) is identified by immunological assay.

38 , The method of claim 35, wherein the one or more proteins identified in step (d) is identified using mass spectrometry,

39. The method of claim 35 , wherein the one or more proteins identified in step (d) is identified by mass-to-charge ratio and retention time.

40. The method of claim 35 , wherein the one or more proteins identified in step (d) is identified by determining, at least in part, the protein's amino acid sequence.

41. The method of claim 35, wherein the one or more proteins identified in step (d) are identified using fluorescence.

42. A kit comprising a volatile buffer and instructions for the use of said volatile buffer as a binding buffer in an immunoaffinity assay.