CN101173926A - Dual quantification method and reagent kit for stable isotope 18 O marked proteome - Google Patents
Dual quantification method and reagent kit for stable isotope 18 O marked proteome Download PDFInfo
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- CN101173926A CN101173926A CNA2006101460116A CN200610146011A CN101173926A CN 101173926 A CN101173926 A CN 101173926A CN A2006101460116 A CNA2006101460116 A CN A2006101460116A CN 200610146011 A CN200610146011 A CN 200610146011A CN 101173926 A CN101173926 A CN 101173926A
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Abstract
The invention provides a new method for double quantifying the protein group marked on the basis of stable oxygen isotope <18>O, which is characterized in that: based on the fact that the N end peptide section and the C end peptide section are generally labeled, and by means of combining the <18>O chemical labeling and enzyme digestion, the quantifying strategy for two <18>O markings of the same sample can be accomplished. The method relates to the reductive alkylation and enzyme digestion of protein, dual-functional reagent modifying and <18>O marking of peptides, multi-dimensional liquid phase chromatogram separation and multistage Tandem Mass Spectrometry quantification and identification. A first mass spectrum peak area is used to quantify; and the identification of the protein is conducted through a second stage mass spectrum and by searching the database. The invention also provides a reagent box for facilitating the implementation of the method.
Description
Technical field
The present invention relates to dual quantitative methods of protein and kit in a kind of proteomics.Comprise a kind of stable isotope that is used for quantitative proteomics
18The O mark is in conjunction with the new method and the kit of the protein of bisgallic acid acid anhydride reagent chemical labeling.
Background technology
Proteomics has become the important content of current life science.The composition of cell protein under the different physiological and pathological conditions and the quantitative or differential protein group research of variation thereof are studied in scale, have become the focus and the difficult point of proteomics research.Accurate quantitative problem in the proteome research has proposed new challenge to present protein group quantivative approach and technology, also more and more is subjected to various countries researchist's attention.
At present, scale protein group quantivative approach mainly contains the colouring method that depends on 2DE and the mass spectrum detection of cold labeling.Because 2-DE can not be high or extremely low to molecular weight, the isoelectric point protein that extremely acid or utmost point alkali and content are low and memebrane protein etc. effectively separate and present, thereby limited its application.The quantitative strategies of introducing stable isotope chemical labeling connexus spectral technology has become the focus of proteomics in recent years.At present, prices such as commercial quantification kit such as isotope affinity tag ICAT, iTRAQ reagent all compare expensive, and method itself also has certain limitation, makes widespread use be restricted.The essential amino acid body internal labeling SILAC method of cold labeling only adapts to the cellular incubation pattern, can not be used for organizing or the quantitative test of body fluid protein group.
Comparatively speaking, the method for enzymatic is introduced when cutting by the protein enzyme
18The O atom, because the labeled reactant step is few, system is simple relatively, therefore receives increasing concern.Protein is at H
2 18Enzymolysis in the O water, but under the effect of enzymatic C-end carboxyl stable bond one or two
18The O atom, but owing to hold carboxyl in conjunction with one or two at C-
18The O atom is at random, and is that pancreatin relies on, and different peptide sections exchanges up
18O atomic time difference makes quantitatively complicated.Protein is existed
18Cut by Lys-N enzyme enzyme in the water of O, but the C-of peptide section holds 1 of carboxyl stable bond under certain conditions
18The O atom.The defective of this method is that the price of Lys-N enzyme is more expensive.In order to address the deficiencies of the prior art, this patent has been invented a kind of based on stable isotope
18The dual quantitative new method of the protein group of O mark.
Summary of the invention
The invention provides a kind of based on stable isotope
18The dual quantitative new method of the protein group of O mark.This method is to be based upon on the basis of N end peptide section and the whole marks of C end peptide section, by
18O chemical labeling and the method that enzyme trimscript note combines can be realized two kinds to same sample
18The quantitative strategies of O cold labeling.The inventor finds to use stable isotope in conjunction with multidimensional liquid chromatography separation means and plural serial stage mass spectrum
18The dual quantitative New Policy of O labelled protein group can be used for improving the accuracy and the reliability of proteomics relative quantification.
A kind of protein group of being used for provided by the invention
18The dual quantitative new method of the protein group of O mark comprises:
(1) opens disulfide bond in the protein molecule with reductive agent, destroy its higher structure; Seal sulfydryl with alkylating reagent, prevent that it from forming disulfide bond again;
(2) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry; Protect with the side chain amino that guanidine radicals reagent is cut the peptide section to enzyme;
(3) the peptide section is carried out chemical modification and processing and carries out mass spectrum quantitative, it is characterized in that the disposal route of described step (3) is:
(a) with one or more bisgallic acid acid anhydride reagent the peptide section is modified, make itself and peptide section generation acetylation coupling reaction;
(b) first amount of the resetting reaction solution that is labeled as bisgallic acid acid anhydride reagent and peptide section coupling reaction is respectively
16O and
18O bicarbonate triethylamine buffer solution; One end generation coupling reaction of bisgallic acid acid anhydride reagent, other end acid anhydrides generation hydrolysis reaction.In the peptide section after the modification, reaction system is at H
2 18Introduced 1 in the peptide section of the bicarbonate triethylamine buffer solution of O configuration
18The O atom.
(c) with two kinds of equivalent
18O and
16Heating made the trypsase deactivation in 10 minutes to the peptide section of O mark through boiling water bath, and equal proportion merges then;
(d) will merge the peptide section with the chromatographic resolution means and separate, analyze with tandem mass spectrum.Carry out the quantitative test of mass spectrometric data and the evaluation of protein.
For further improving accuracy of analysis, this method can also be proceeded second amount of resetting, and the steps include:
(e) with half reaction solution in (b), 37 ℃, hatch 18h.Reaction system is
18The C of the modified peptides section of O end two of c-terminus under the effect of tryptose enzymatic
16The O atom is exchanged for
18O.
(f) with two kinds of equivalent
18O and
16Heating made the trypsase deactivation in 10 minutes to the peptide section of O mark through boiling water bath, and equal proportion merges then;
(g) will merge the peptide section with the chromatographic resolution means and separate, analyze with tandem mass spectrum.Carry out the evaluation that second of mass spectrometric data resets component analysis and protein.
Wherein, the described bisgallic acid acid anhydride of step (a) reagent is preferably is with amino bisgallic acid acid anhydride in the molecule, aromatic amine bisgallic acid acid anhydride, or the compound of the active spacer group of isothiocyanic acid base bisgallic acid acid anhydride.Be preferably diethylenetriamine pentaacetic acid bisgallic acid acid anhydride especially.
The described stable isotope of step (b) is
18O, buffer solution is respectively
16The bicarbonate triethylamine buffer salt of O water configuration and
18The bicarbonate triethylamine buffer salt solution of O water configuration.
Step (d) and (g) the preferred LC-ESI-MS/MS electro-spray ionization of described mass spectrum tandem mass spectrum, or the MALDITOF/TOF ground substance assistant laser flight time tandem mass spectrum that dissociates more preferably use LC-MS; Database search method is preferably adopted in the parsing of mass spectrometric data and the evaluation of protein.
This method can be carried out the modification of bisgallic acid acid anhydride reagent to the peptide section that the protein enzyme of two kinds of different physiological statuss is cut generation; Use dual again
18The O stable isotope carries out mark, and two kinds of labeling methods obtain
18O with
16The peptide section of O mark equal proportion is respectively mixed, and realizes the dual quantitative of protein group by twice multi-dimensional chromatograph separation and tandem mass spectrum assay determination.Heavy first
18Introduced 1 in the O labeling method
18The O atom is in the one-level mass spectrogram
16O and
18The a pair of peptide section mass number difference of O mark is 2Da (single electric charge), does not exist
18O and
16Exchange between the O atom; Heavy second
183 have been introduced in the O labeling method
18The O atom is in the one-level mass spectrogram
16O and
18The a pair of peptide section mass number difference of O mark is 6Da (single electric charge), has avoided the overlapping of isotopic peak.
16O and
18The ionic strength ratio of a pair of peptide section of O mark, the relative scale of just corresponding two kinds of comparison proteins, the mass number difference that doubly charged peptide section is right be proper mass count difference 1/2nd, the rest may be inferred.Carry out database retrieval by second order ms and come identification of protein.
Bisgallic acid acid anhydride reagent is in the molecule or is with amino bisgallic acid acid anhydride, or band aromatic amine bisgallic acid acid anhydride, or the compound of the active spacer group of band isothiocyanic acid base bisgallic acid acid anhydride.As diethylenetriamine pentaacetic acid bisgallic acid acid anhydride (bicyclic anhydride diethylenetriamine N, N, N '; N ", N "-pentaacetic dianhydride, DTPAA) be bisgallic acid acid anhydride reagent; its end can with the amido coupling of peptide section, hydrolysis reaction can take place in the other end.This reagent is cheap.This method is with at present traditional
18The method of O enzyme trimscript note is compared, and does not have
16O and
18Exchange between the O atom can reduce the error of quantitative experiment, improves quantitative accuracy.A kind of labelling strategies in addition is to set up on the basis of first kind of labeling method.Because the compatibility of reaction system, having set up mass number difference is the quantivative approach of 6Da, this method and traditional
18The method that the O enzyme is cut is compared, and has avoided the overlapping of isotopic peak.
On the other hand, the present invention also provides and has been used for proteomics
18The dual quantitative kit of O cold labeling protein, the component of this kit comprises:
(a) one or more can with the bisgallic acid acid anhydride reagent of peptide section coupling;
(b) coupling reaction
16O water and
18The bicarbonate triethylamine buffer solution of O water configuration;
(c) can carry out the chromatographic column of peptide section chromatographic resolution.
Wherein, be with amino bisgallic acid acid anhydride in the preferred molecule of component (a), aromatic amine bisgallic acid acid anhydride, or the compound of the active spacer group of isothiocyanic acid base bisgallic acid acid anhydride; The preferred reversed-phase column of component (c) or ion exchange column, more preferably on-line coupling chromatographic column.
As is known to the person skilled in the art, above component only is schematic.Use for convenient, this kit also can comprise one or more in the following component: be used for opening the reductive agent of protein molecule disulfide bond, as dithiothreitol (DTT) (DTT) and three carboxyethyl phosphines (TCEP); The sealing sulfydryl prevents that it from forming the alkylating reagent of disulfide bond again, as iodoacetamide and iodoacetic acid; The protein digestibility agent is as trypsase; The side chain amido protecting agent is as the O-methyl-isourea; Can contain the buffer system that one or more are used for peptide section chromatographic resolution in addition; The operation instruction of state administrative organs's approval etc.
New method of the present invention and kit be applicable to the protein difference expression to the tissue of two kinds of different conditions or cell carry out relative quantitatively, this method is quick, and is easy to use.Difference on can the protein expression level of the following two kinds of samples of more different simultaneously physiological and pathological conditions, thereby the easy differential protein that screens this physiological and pathological condition of sign rapidly and accurately, and then the searching biomarker, for medical diagnosis on disease and drug screening provide a kind of analysis tool simply fast.Quantification of protein and qualitative analysis can be finished in an experimental period synchronously.Be specially adapted to carry out in biology, medical science and the field of pharmacology differential protein group or comparison protein group analysis.
This method is a protein difference expression relative quantitative assay instrument, reduces the cost of relative quantification in the proteomics greatly.And reaction conditions is gentle, simple to operate relatively, realizes easily.Quantification of protein and qualitative analysis can be finished in an experimental period synchronously.This method is to be based upon on the basis of N end peptide section and the general mark of C end peptide section, by
18O chemical labeling and the method that enzyme trimscript note combines can be realized two kinds to same sample
18The quantitative strategies of O cold labeling.Method of the present invention and kit have practicality widely.Purposes includes but not limited to: the proteome research that carries out various animals and plants and microorganism in fields such as medical science, pharmacy, agricultural and animal husbandry.
Description of drawings
Fig. 1. be the experiment flow synoptic diagram of this method
Fig. 2. myoglobins peptide section
18O-DTPA-VEADIAGHGQEVLIR with
16The mixed one-level mass spectrogram of O-DTPA-VEADIAGHGQEVLIR equal proportion is first weight
18The O mark is quantitative, is that the ratio that the actual ratio of the area of 1981.6384 and 1983.6417 isotopic peak is carried out the corresponding peptides section is calculated according to m/z in the drawings.Calculating is with reference to list of references (Sekhar Rao K.C., Carruth R.T., and Miyagi M., Proteolytic
18O Labeling by Peptidyl-LysMetalloendopeptidase for Comparative Proteomics, Journal of Proteome Research, 2005,4,507-514).The ratio that calculates is 1: 1.02, and is approaching with theoretical value 1: 1.
Fig. 3. myoglobins peptide section
18O-DTPA-VEADIAGHGQEVLIR-2
18O with
16O-DTPA-VEADIAGHGQEVLIR-2
18The mixed one-level mass spectrogram of O equal proportion is second weight
18The O mark is quantitative, is that the area 29265.2852 of 1982.1433 and 1988.1532 isotopic peak and ratio that 28576.7188 actual ratio is carried out the corresponding peptides section are calculated according to m/z in the drawings.The ratio that calculates is 1: 1.02, and is approaching with theoretical value 1: 1.
Fig. 4 .A figure is a myoglobins peptide section
18The second order ms figure of O-DTPA-VEADIAGHGQEVLIR; B figure is a myoglobins peptide section
18O-DTPA-VEADIAGHGQEVLIR-2
18The second order ms figure of O.The secondary fragment peak that the secondary fragment peak of the parent ion that obtains among A and the B figure is the continuous parent ion that obtains is continuous y ion (from y1 to y15), makes collection of illustrative plates simplify greatly, can significantly improve the reliability that the peptide section is identified.
Embodiment
The following examples will be further explained the present invention, but the present invention is not limited only to these embodiment, the scope that these embodiment do not limit the present invention in any way.Some change that those skilled in the art is made within the scope of the claims and adjust also should be thought and belongs to scope of the present invention.
The white dual quantitative mark of embodiment 1 horse cardiac muscle red eggs
1.1 it is white to get 0.1mg horse cardiac muscle red eggs, is dissolved in the bicarbonate triethylamine buffer solution of 100 μ L 50mM pH7.0.5 minutes protein denaturations of 95 ℃ of heating.There is not cysteine residues in the amino acid sequence of myoglobins, so do not need the reductive alkylation step.Add 2ug trypsase, 37 ℃ of enzymes were cut 2 hours.Add 2ug trypsase again, 37 ℃ of enzymes were cut 10 hours.Enzyme is cut the myoglobins peptide section that obtains is equally divided into four parts, wherein two parts of boiling water baths heating ten minutes then ice bath cool off rapidly, make the trypsase deactivation.Then with remaining two parts bone dries in the vacuum drier together.In the pressed powder that obtains, getting deactivation of a copy of it pancreatin and a pancreatin does not have the sample of deactivation to be dissolved in 25 μ L
18In the bicarbonate triethylamine buffer solution of the 50mM pH7.0 of O water configuration.In remaining two parts of bicarbonate triethylamine buffer solution that are dissolved in 25 μ L50mM pH7.0.Then four parts of potpourris are joined respectively in the pressed powder of 360 μ g diethylenetriamine pentaacetic acid bisgallic acid acid anhydrides, behind the violent whirlpool shake 1min, normal temperature was placed 1 hour.The solution of trypsase deactivation is mixed according to 1: 1 ratio.The direct LC-Packing burble point of biased sample target, MALDI-TOF/TOF detects evaluation.Trypsase does not have the solution of deactivation, is positioned in 37 ℃ of water-baths, hatches 18h.Mix according to 1: 1 ratio then, biased sample is before the LC-MS isolation identification, and the boiling water bath heating made the abundant inactivation of trypsase in 10 minutes.
1.2 respectively getting the mixed sample of 0.6 μ L receives upgrading capillary liquid chromatography system with U.S. Dionex-LC Packings and separates.The kapillary reverse-phase chromatographic column is 15cm * 75 μ m i.d. silica glass tubes, interior dress Vydac C18, and diameter 5 μ m, the aperture is
Filler.Pre-column is 5mm * 320 μ m i.d., and filler is PepMap C18, diameter 5 μ m, and the aperture is
Mobile phase A: water/acetonitrile (95/5, V/V), contain 0.1% trifluoroacetic acid, Mobile phase B: acetonitrile/water (95/5, V/V), contain 0.1% trifluoroacetic acid, shunting back flow velocity is 200nl/min, detects wavelength 214nm.Separate and adopt nonlinear gradient: 100%A, 20min, 0%～20%B, 10min; 20%～60%B, 40min; 60%～100%B, 10min; 100%B, 10min; 100%B～100%A, 5min; 100%A, 15min.Behind the wash-out 30min, the automatic bleeding point target of online cut, treat the cut bone dry after, every 5mg/ml a-cyano group-4-hydroxycinnamic acid matrix solution of adding 0.5 μ l carries out the MALDI-TOF/TOF mass spectrophotometry after to be dried.
The substance assistant laser desorpted ionized flight time tandem mass spectrometer of the 4700Proteomics Analyzer of American AB I company (MALDI-TOF/TOF).MS-1KV reflective-mode, accelerating potential 20KV, sweep limit 700-3500m/z are used in the collection of one-level mass spectrometric data.Second order ms The data MS/MS-1KV reflective-mode selects signal to noise ratio (S/N ratio) greater than 20 peak from the one-level spectrogram, and basis signal intensity is selected the analysis of connecting of 8 parent ions from high to low.
Retrieval software is Mascot v1.9, and database is Swiss-prot.Search parameter comprises that the variable mass number that is modified at the N end mark O-DTPA label of peptide section adds 376.1Da, at the N of peptide section end mark
18The mass number of O-DTPA label adds 378.1Da; The peptide segment mark is signed to the mass number of O-DTPA-20 adds 379.13Da in second kind of labeling method, and the peptide segment mark is signed and is
18O-DTPA-2
18The mass number of O adds 385.13Da.
1.3 can obtain the corresponding peak area ratio of different peptide sections from the one-level mass spectrogram.As Fig. 2 is myoglobins peptide section
18O-DTPA-peptide with
16After the O-DTPA-peptide equal proportion is mixed
18O-DTPA-VEADIAGHGQEVLIR with
16The one-level mass spectrogram of O-DTPA-VEADIAGHGQEVLIR.Real peak area ratio computing method reference literature (Sekhar RaoK.C., Carruth R.T., and Miyagi M., Proteolytic
18O Labeling by Peptidyl-LysMetalloendopeptidase for Comparative Proteomics, Journal of Proteome Research, 2005,4,507-514).The ratio that calculates is 1: 1.02, and is approaching with theoretical value 1: 1.Fig. 3 is a myoglobins peptide section
18O-DTPA-peptide-2
18O with
16O-DTPA-peptide-2
18After the O equal proportion is mixed
18O-DTPA-VEADIAGHGQEVLIR-2
18O with
16O-DTPA-VEADIAGHGQEVLIR-2
18The one-level mass spectrogram of O.The ratio that calculates is 1: 1.02, and is approaching with theoretical value 1: 1.And two kinds of experimental result unanimities that method obtains can be verified mutually.Quantitative accuracy and reliability have been improved greatly.Fig. 4 A figure is a myoglobins peptide section
18The second order ms figure of O-DTPA-VEADIAGHGQEVLIR; B figure is a myoglobins peptide section
18O-DTPA-VEADIAGHGQEVLIR-2
18The second order ms figure of O.As can be seen from the figure in their second order spectrum, obtain continuous y series ion, made collection of illustrative plates simplify greatly, can significantly improve the reliability that the peptide section is identified.
2 six kinds of standard protein potpourris of embodiment dual quantitatively
2.1 six kinds of standard protein potpourris are configured (potpourri A and B, μ g/mL) according to following ratio: beta-casein (10,10) all available from U.S. Sigma company; Beta lactoglobulin (10,40); Myoglobins (60,20); Bovine serum albumin(BSA) (40,20); Lysozyme (50,50); Transferrins (30,60).Protein mixture is dissolved in 50 μ L pH7.0,50mM bicarbonate triethylamine solution, 95 ℃ of heat denatured 5min.Add 50mM reductive agent three carboxyethyl phosphorus 2 μ L, hatch 1h for 60 ℃.Add 2 μ L 250mM alkylating reagent iodoacetamides then, placed 10 minutes the dark place, and proportionally (1: 20w/w) add trypsase, 37 ℃, 18h.Enzyme is cut the protein peptide section solution for vacuum drying that obtains, add
18The bicarbonate triethylamine buffer solution dissolving of O water configuration is with excessive 5 times bisgallic acid acid anhydride pressed powder room temperature reaction 30min.The peptide section N end that obtains is marked with one
18The O atom.This potpourri is equally divided into two parts, and the heating of a copy of it boiling water bath made the trypsase deactivation in 10 minutes, carried out the LC-MS isolation identification then.A in addition solution is positioned in 37 ℃ of water-baths, hatches 18h.Before the LC-MS isolation identification, the boiling water bath heating made the abundant inactivation of trypsase in 10 minutes.Mix according to 1: 1 ratio respectively.The direct LC-Packing burble point of biased sample target, MALDI-TOF/TOF detects evaluation.Trypsase does not have the solution of deactivation, is positioned in 37 ℃ of water-baths, hatches 18h.Mix according to 1: 1 ratio then, biased sample is before the LC-MS isolation identification, and the boiling water bath heating made the abundant inactivation of trypsase in 10 minutes.
2.2 respectively getting the mixed sample of 0.6 μ L receives upgrading capillary liquid chromatography system with U.S. Dionex-LC Packings and separates.The kapillary reverse-phase chromatographic column is 15cm * 75 μ m i.d. silica glass tubes, interior dress Vydac C18, and diameter 5 μ m, the aperture is
Filler.Pre-column is 5mm * 320 μ m i.d., and filler is PepMap C18, diameter 5 μ m, and the aperture is
Mobile phase A: water/acetonitrile (95/5, V/V), contain 0.1% trifluoroacetic acid, Mobile phase B: acetonitrile/water (95/5, V/V), contain 0.1% trifluoroacetic acid, shunting back flow velocity is 200nl/min, detects wavelength 214nm.Separate and adopt nonlinear gradient: 100%A, 20min, 0%～20%B, 10min; 20%～60%B, 40min; 60%～100%B, 10min; 100%B, 10min; 100%B～100%A, 5min; 100%A, 15min.Behind the wash-out 30min, the automatic bleeding point target of online cut, treat the cut bone dry after, every 5mg/ml a-cyano group-4-hydroxycinnamic acid matrix of adding 0.5 μ l is carried out the MALDI-TOF/TOF mass spectrophotometry after to be dried.
The substance assistant laser desorpted ionized flight time tandem mass spectrometer of the 4700Proteomics Analyzer of American AB I company (MALDI-TOF/TOF).MS-1KV reflective-mode, accelerating potential 20KV, sweep limit 700-3500m/z are used in the collection of one-level mass spectrometric data.Second order ms The data MS/MS-1KV reflective-mode selects signal to noise ratio (S/N ratio) greater than 20 peak from the one-level spectrogram, and basis signal intensity is selected the analysis of connecting of 8 parent ions from high to low.
Retrieval software is Mascot v1.9, and database is Swiss-prot.Search parameter comprises that the variable mass number that is modified at the N end mark O-DTPA label of peptide section adds 376.1Da in first heavy label, at the N of peptide section end mark
18The mass number of O-DTPA label adds 378.1Da.The peptide segment mark is signed to the mass number of O-DTPA-20 adds 376.1Da in the second heavy label method, and the peptide segment mark is signed and is
18O-DTPA-2
18The mass number of O adds 382.1Da.
2.3 in the result of mass spectroscopy, all MSMS data have identified this six standard proteins by database retrieval, table 1 is six kinds of standard proteins
18The dual quantitative result of O labelled protein group.Experimental result is as shown in table 1.As can be seen from the table,
18Two kinds of quantitative strategies experimental errors of O mark all in 20%, prove that this method has the value of practical application in proteomics.
Table 1 standard protein
18The dual quantitative result of O labelled protein group
Claims (12)
1. one kind is used for the dual quantitative stable isotope of protein group
18The method of O labelled protein comprises:
(1) opens disulfide bond in the protein molecule with reductive agent, destroy its higher structure; Seal sulfydryl with alkylating reagent, prevent that it from forming disulfide bond again;
(2) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry; Protect with the side chain amino that reagent is cut the peptide section to enzyme;
(3) the peptide section is carried out chemical modification and processing and carries out mass spectrum quantitative;
The disposal route that it is characterized in that described step (3) is:
(a) with one or more bisgallic acid acid anhydride reagent the peptide section is modified, make itself and peptide section generation acetylation coupling reaction;
(b) first amount of the resetting reaction solution that is labeled as bisgallic acid acid anhydride reagent and peptide section coupling reaction is respectively
16O and
18O bicarbonate triethylamine buffer solution; One end generation coupling reaction of bisgallic acid acid anhydride reagent, other end acid anhydrides generation hydrolysis reaction.In the peptide section after the modification, reaction system is at H
2 18Introduced 1 in the peptide section of the bicarbonate triethylamine buffer solution of O configuration
18The O atom.
(c) with two kinds of equivalent
18O and
16Heating made the trypsase deactivation in 10 minutes to the peptide section of O mark through boiling water bath, and equal proportion merges then;
(d) will merge the peptide section with the chromatographic resolution means and separate, analyze with tandem mass spectrum.Carry out the quantitative test of mass spectrometric data and the evaluation of protein.
2. method according to claim 1 is characterized in that also comprising following second amount of the resetting step:
(e) with half reaction solution in (b), 37 ℃, hatch 18h.Reaction system is
18The C of the modified peptides section of O end two of c-terminus under the effect of tryptose enzymatic
16The O atom is exchanged for
18O.
(f) with two kinds of equivalent
18O and
16Heating made the trypsase deactivation in 10 minutes to the peptide section of O mark through boiling water bath, and equal proportion merges then;
(g) will merge the peptide section with the chromatographic resolution means and separate, analyze with tandem mass spectrum.Carry out the evaluation that second of mass spectrometric data resets component analysis and protein.
3. method according to claim 1 and 2 is characterized in that the described bisgallic acid acid anhydride of step (a) reagent is to be with amino bisgallic acid acid anhydride in the molecule, aromatic amine bisgallic acid acid anhydride, or the compound of the active spacer group of isothiocyanic acid base bisgallic acid acid anhydride.
4. method according to claim 1 and 2 is characterized in that the described anhydride reagent in pairs of step (a) is a diethylenetriamine pentaacetic acid bisgallic acid acid anhydride.
5. method according to claim 1 and 2 is characterized in that the described stable isotope of step (b) is
18O, buffer solution is respectively
16The bicarbonate triethylamine buffer salt of O water configuration and
18The bicarbonate triethylamine buffer salt solution of O water configuration.
6. method according to claim 1 and 2, it is characterized in that step (d) and (g) described mass spectrum be LC-ESI-MS/MS electro-spray ionization tandem mass spectrum, or the MALDI TOF/TOF ground substance assistant laser flight time tandem mass spectrum that dissociates.
7. method according to claim 1 and 2, it is characterized in that step (d) and (g) authentication method of the parsing of described mass spectrometric data and protein for using database search.
8. method according to claim 1 is characterized in that the disposal route of described step (3) is:
(a) with diethylenetriamine pentaacetic acid bisgallic acid acid anhydride the peptide section is modified, make itself and peptide section generation acetylation coupling reaction;
(b) first amount of the resetting reaction solution that is labeled as diethylenetriamine pentaacetic acid bisgallic acid acid anhydride and peptide section coupling reaction is respectively
16The bicarbonate triethylamine buffer salt of O water configuration and
18The bicarbonate triethylamine of O water configuration
18The O buffer salt;
(c) with two kinds of equivalent
18O and
16Heating made the trypsase deactivation in 10 minutes to the peptide section of O mark through boiling water bath, and equal proportion merges then;
(d) will merge the peptide section with the chromatographic resolution means and separate, analyze with tandem mass spectrum.Carry out the quantitative test of mass spectrometric data and the evaluation of protein.
(e) second amount of resetting is labeled as, and with half reaction solution in (b), 37 ℃, hatches 18h.Reaction system is
18The C of the modified peptides section of O end two of c-terminus under the effect of tryptose enzymatic
16The O atom is exchanged for
18O.
(f) with two kinds of equivalent
18O and
16Heating made the trypsase deactivation in 10 minutes to the peptide section of O mark through boiling water bath, and equal proportion merges then;
(g) will merge the peptide section with the chromatographic resolution means and separate, analyze with tandem mass spectrum.Carry out the quantitative test of mass spectrometric data and the evaluation of protein.
9. method according to claim 1 and 2, the disposal route that it is characterized in that described step (3) are two kinds of same sample
18The O labeling method can realize the dual quantitative of two kinds of methods.
10. one kind is used for the quantitative proteomics stable isotope
18The kit of O labelled protein group is characterized in that the component of this kit comprises:
(a) one or more can with the bisgallic acid acid anhydride reagent of peptide section coupling;
(b) H of coupling reaction
2 16O and H
2 18The bicarbonate triethylamine buffer solution of O;
(c) can carry out the chromatographic column of peptide section chromatographic resolution.
11. kit according to claim 10 is characterized in that the described bisgallic acid acid anhydride of component (a) reagent is to be with amino bisgallic acid acid anhydride in the molecule, aromatic amine bisgallic acid acid anhydride, or the compound of the active spacer group of isothiocyanic acid base bisgallic acid acid anhydride.
12. kit according to claim 10 is characterized in that the described chromatographic column of component (c) is reversed-phase column or ion exchange column.
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