CN110297056A

CN110297056A - A kind of preparation and application of isobar tagging reagents

Info

Publication number: CN110297056A
Application number: CN201910582411.9A
Authority: CN
Inventors: 李舒伟
Original assignee: Nanjing Pelijian Biotechnology Co Ltd
Current assignee: Nanjing Pelijian Biotechnology Co Ltd
Priority date: 2019-06-29
Filing date: 2019-06-29
Publication date: 2019-10-01
Also published as: CN111024867A

Abstract

The invention discloses a kind of isobar tagging reagents, the structure feature of isobar tagging reagents includes following formula: reporter group-balance group-activated group, isobar tagging reagents include 10 identical tagging reagents of chemical structure, different location in each tagging reagents contains 13C or 15N isotope, and isobar tagging reagents include: T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N, T-117C, T-118C, T-119；The method for preparing the isobar tagging reagents is also disclosed simultaneously, is also disclosed using the isobar tagging reagents to polypeptide quantitative analysis method.IBT-10PLEX in the present invention uses novel chemical structure, reduces the difficulty of preparation, improves yield, so as to reduce the cost of product, expands the use scope of isobar labelling method.

Description

A kind of preparation and application of isobar tagging reagents

Technical field

The present invention relates to biomolecule analysis reagent fields, are especially used for biomolecule quantitative analysis, particularly for A kind of preparation of quantitative isobar tagging reagents of the isobar (isobaric) containing 10 labels of proteomics and Using.

Background technique

It mainly include unmarked analytic approach and label currently based on the quantitative approach of the proteomics of mass-spectrometric technique detection Method, and labelling method is divided into based on the method that first mass spectrometric (MS1) is quantitative and second order ms (MS2) are quantitative, wherein isobar Isotope labelling belongs to the analysis method based on MS2.

Compared with unmarked analytic approach and MS1 labelling method, MS2 labelling method advantage is:

1) analysis time of Bulk Samples can be substantially reduced.

2) it is mixed in advance before liquid phase series connection (LC-MS/MS) mass spectral analysis due to the different samples of isobar label It closes, so that whole samples are completed in a LC-MS/MS analysis, effectively reduces different samples in different LC-MS/MS Systematic error.Isobar labeling method can reduce what different samples generated in not herewith measurement to the maximum extent Difference solves the problems, such as the unavoidable repeatability in the analysis method that no label marks.

3) multiple samples mix after isobar reagent label, same polypeptide contained in different samples point Son is merged together, and is equivalent to and is enhanced sample concentration, and corresponding MS/MS fragment signal becomes stronger, makes the sensitive of detection Degree greatly improves.

4) based on the labeling method of MS1, the amino acid containing stable isotope labeling is generally added in cell culture medium (SILAC labelling method).In contrast, isobar MS2 labeling method is eased, reduce the former answering in chromatographic isolation Polygamy is more applicable for the comparative analysis of multiple samples.

5) arginine or bad ammonia element of the quantitative approach dependent on cellular uptake containing stable isotope based on SILAC label close At albumen, and then contain the protein of labeled amino acid containing these again.This method only may be implemented in the cell of culture, But in bacterium, animal tissue is difficult to apply in the analysis of human sample and realize.In contrast, isobar isotope MS2 is marked Note will not then be limited by samples sources.

Obviously, isobar labelling method is one of most popular method during proteomics is quantitative.However, existing on the market Commercialized isobar labelled reagent valuable product become many especially for the sample for largely needing to mark The financial burden in laboratory and it is maximum apply obstacle.

Summary of the invention

To solve the above problems, the invention discloses a kind of isobars for MS2 fragment label to contain 10 labels The preparation and application of reagent (IBT-10PLEX).Reagent contain 10 kinds of label labels can different samples of 10 kinds of label simultaneously, it is complete The full polypeptide quantitative analysis for being suitable for multiple proteins group.

In order to reach the goals above, the invention provides the following technical scheme:

Isobar tagging reagents (IBT-10PLEX reagent): shear key and isotopic tag can be contained comprising MS/MS Polypeptide can be marked and quantified with the group of polypeptides reactive.Its structure includes following formula: reporter group-balance group-work Change group, wherein reporter group and balance group, which pass through MS/MS, can shear key connection.IBT-10PLEX reagent labeling polypeptide it is anti- Polypeptide after answering and marking cracks the structure such as following figure of the reporter group of formation in tandem mass spectrum.

A whole set of reagent includes 10 identical tagging reagents of chemical structure, the different location in each tagging reagents Contain 13C or 15N isotope.Therefore the quality of reporter group is different, but the different quality of reporter group is balanced base Group is compensated.Reporter group+balance group quality in each reagent is identical.Reporter group and balance group pass through can Shearing key connection, shear key are preferentially broken in MS/MS, generate a series of reporter group ions, be respectively provided with different molecules Formula and quality, the specific molecular formula and accurate mass of reporter group ion are as follows:

Its corresponding chemical structure is as follows, and wherein asterisk label represents the position of isotope 15N and 13C.

Wherein in the range of 114 to 119Da, the reagent contains independently selected from 13C the quality of the reporter group And/or the isotope atom combination of the different number and location of 15N, and identification and analysis is carried out by mass spectrum；Identification and analysis step It is as follows:

1) liquid chromatogram separation is carried out using nanometer reverse-phase chromatographic column；

2) it is analyzed using peptide identification of the Orbitrap Q Exactive high-resolution mass spectrometer to label；

3) it is at least the 5% of peak-peak using the signal strength of the signal reports group ion of quantitative analysis.

IBT-10PLEX reagent can directly be marked 10 kinds of polypeptide samples.It is mixed for LC-MS/ after the completion of label MS analysis.Since reporter group and balance group can shear key connection by mass spectrum MS/MS, it is cut off in Tandem Mass Spectrometry Analysis Be formed by report ion difference in quality can be analyzed with high resolution mass spectrum after for quantitative.Further, since IBT- 10PLEX contains only 13C and 15N isotope, and without containing another common isotope deuterium, avoid relevant color caused by deuterium Spectral displacement, it is ensured that dosing accuracy.

The invention also discloses a kind of methods for synthesizing 7 kinds of isotopic tags, using the raw material β-for containing different isotopes Ala-OBzl, Boc-Gly-OH, propionic aldehyde synthesize 7 kinds of isotopic tags T-114, T-115N, T-115C, T- according to following methods 116N,T-116C,T-117N,T-118N；

Specific step is as follows:

1) 1 β-Ala-OBzl.HCl of compound and compound 2Boc-Gly-OH are coupled to compound 3；

2) compound 3 sloughs Boc group in acid condition, forms compound 4；

3) compound 4 and propionic aldehyde form compound 5 under reducing agent NaCNBH3 effect；

4) compound 5 and hydrogen slough Bzl blocking group under the catalytic action of palladium carbon and form compound 6；

5) compound 6 forms the IBT reagent compound 7 of activation with NHS under the action of dehydrating agent EDC；

Process route is as follows:

The invention also discloses the methods of 3 kinds of isotopic tags of synthesis, using the raw material β-Ala- for containing different isotopes Obzl, Boc-Gly-OH, propionic aldehyde synthesize 3 kinds of isobar tagging reagents, including T-117C, T-118C, T- according to following methods 119；Specific step is as follows:

1) 2-Nosyl-Gly-OH (compound 8) reacts to form compound 9 with iodopropane；

2) compound 9 is deprotected under the conditions of 2 mercapto ethanol and potassium hydroxide and forms compound 10；

3) compound 11 is formed plus Boc blocking group on the second level amino of compound 10；

4) β-Ala-OBzl.HCl (compound 1) and compound 11 are coupled to compound 12；

5) compound 12 sloughs Boc group in acid condition, forms compound 13；

6) compound 13 and propionic aldehyde form compound 5 under reducing agent (NaCNBH3) effect；

7) compound 5 and hydrogen slough Bzl blocking group under the catalytic action of palladium carbon and form compound 6；

8) compound 6 forms the IBT reagent compound 7 of activation with NHS under the action of dehydrating agent EDC；

The application of IBT-10PLEX reagent: IBT-10PLEX reagent is used for polypeptide quantitative analysis, and key step includes label, Molecular fragment, quantitative three steps.After most 10 polypeptide samples can be marked respectively by IBT-10PLEX reagent, it is blended in one It rises, is then separated on liquid chromatogram LC, and then generate fragments characteristic peak and reagent reporter group in MS/MS, according to spy The intensity for levying peak and reporter group carries out qualitative, quantitative analysis to polypeptide.

Wherein, polypeptide is prepared by the following:

(1) from the albumen of cell extraction

(2) pass through trypsin digestion

(3) desalination obtains mixtures of polypeptides

Wherein polypeptide is obtained from cell culture or other samples.

The preparation of sample may come from the protein ingredient extracted in the cell or other biological sample of culture.

The invention also discloses a kind of isobar tagging reagents, wherein isobar tagging reagents equally contain 13C and The equivalent dystopy isotopic tag of 15N, the isobar tagging reagents include following structure A, structure B, structure C, structure D or The one or more of them of person's structure E:

The invention has the following beneficial effects:

IBT-10PLEX in the present invention uses novel chemical structure, reduces the difficulty of preparation, improves yield, So as to reduce the cost of product, expand the use scope of isobar labelling method.

The present invention has intrinsic efficient, high-throughput, the highly sensitive feature of all isobar reagents, while improving it The defect of his similar reagent has low cost.

Reagent of the invention contain 10 kinds of label labels can different samples of 10 kinds of label simultaneously, be completely suitable for a variety of eggs The polypeptide quantitative analysis of white matter group.

Detailed description of the invention

Influence datagram of the ratio of Fig. 1 (A) IBT-10PLEX reagent and peptide to labeling effciency；

Fig. 1 (B) IBT-10PLEX reagent marks influence datagram of the reaction time to labeling effciency；

Optimal LC-MS/MS mass spectral analysis condition-point needed for Fig. 2 (A): IBT-10PLEX labelled protein quantitative analysis The influence datagram that resolution distinguishes reporter group ion；

Optimal LC-MS/MS mass spectral analysis condition-NCE needed for Fig. 2 (B): IBT-10PLEX labelled protein quantitative analysis (orthogonal collision energy) is identified IBT labeling polypeptide and the influence datagram of IBT report ionic strength；

Fig. 3 (A): the datagram of the accuracy-IBT-10PLEX labeling effciency of IBT-10PLEX reagent quantitative；

Fig. 3 (B): the data of the quantitative linearity scope of the accuracy-IBT-10PLEX label of IBT-10PLEX reagent quantitative Figure；

Fig. 3 (C): the accuracy-IBT-10PLEX label of IBT-10PLEX reagent quantitative is opposite to the sample of different proportion Quantitative accuracy data figure；

Fig. 3 (D): the accuracy of IBT-10PLEX reagent quantitative-complex sample background marks IBT-10PLEX quantitative Influence degree datagram

Fig. 4 (A): IBT-10PLEX reagent is for phosphoeptide in analysis-analysis HeLa cell of phosphoeptide in HeLa cell Flow chart；

Fig. 4 (B): IBT-10PLEX reagent is for analysis-identification and quantification phosphoeptide of phosphoeptide in HeLa cell Incremental data figure；

Fig. 4 (C): analysis-of the IBT-10PLEX reagent for phosphoeptide in HeLa cell becomes in different range relative abundance The quantity of the phosphoeptide of change；

Fig. 4 (D): analysis-representativeness phosphoeptide of the IBT-10PLEX reagent for phosphoeptide in HeLa cell is relatively rich Spend the curve changed over time.

Specific embodiment

With reference to the accompanying drawings and detailed description, the present invention is furture elucidated, it should be understood that following specific embodiments are only For illustrating the present invention rather than limiting the scope of the invention.

The invention discloses a kind of isobar tagging reagents IBT-10PLEX reagent, isotopic tag reagent includes 10 The identical tagging reagents of chemical structure, the different location in each tagging reagents contain 13C or 15N isotope, same to position Plain tagging reagents include: T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N, T-117C, T-118C, T-119.Isotopic tag reagent include MS/MS can shear key and containing isotopic tag can with the group of polypeptides reactive, Polypeptide is marked and is quantified；The structure of the isotopic tag reagent includes following formula: reporter group-balance group-activation Group, wherein reporter group and balance group, which pass through MS/MS, can shear key connection, and activated group reacts to be formed with the amino of polypeptide Stable amido bond；Reporter group+balance group quality in each reagent is identical；Contain isotopic tag reagent Concrete structure formula is as follows:

Wherein, T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N；

Specific step is as follows:

2) compound 3 sloughs Boc group in acid condition, forms compound 4；

Process route is as follows:

T-117C, T-118C, T-119 use raw material β-Ala-Obzl, Boc-Gly-OH, the third containing different isotopes Aldehyde, the specific steps are as follows:

1) 2-Nosyl-Gly-OH (compound 8) reacts to form compound 9 with iodopropane；

4) β-Ala-OBzl.HCl (compound 1) and compound 11 are coupled to compound 12；

5) compound 12 sloughs Boc group in acid condition, forms compound 13；

Process route is as follows:

T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N, T-117C, T-118C, T-119's Shown in specific synthetic route following examples 1 and embodiment 2, of course, T-115N, T-115C, T-116N, T-116C, T- It is 117N, T-118N, consistent with the route of T-114, it is T-117C, T-118C, consistent with the route of T-119.

The synthesis of embodiment 1:T-114, synthetic route are as follows:

The synthesis of T-114, asterisk represents 13C or 15N on corresponding position in synthetic route.

Compound 1 (β-Ala-OBzl.HCl, 13C3,15N, 215mg, 1mmol) and compound 2 (Boc-Gly-OH, 1- 13C, 175mg, 1mmol) it is dissolved in 20mL methylene chloride, it is added EDC.HCl (229mg, 1.2mmol), reaction is protected in argon gas Shield is lower to be carried out 20 hours, and reaction solution is washed three times with 25mM dilute hydrochloric acid 30mL, is removed, is obtained with Rotary Evaporators after organic phase is dry 283mg oily compound 3 (84% yield).EtOAc solution (1M) stirring 2 that 50mL HCl is added in compound 3 (283mg) is small When, the white precipitate of appearance is collected by filtration to obtain 205mg compound as white solid 4 (90% yield).Compound 4 (205mg) is molten Solution is added propionic aldehyde (162 μ L, 130mg) in 20mL methanol, and 2 hours of 45mg sodium cyanoborohydride reaction are then added.With rotation Turn evaporimeter and remove methanol, crude product methylene chloride: ethyl acetate (9:1) purifies obtain oily compound 5 on a silica gel column (183mg, 76% yield).Compound 5 is dissolved in 20mL methanol, after 20mg Pd/C is added, reacts one under hydrogen balloon effect A hour.It removes solvent and obtains compound as white solid 6 (125mg, 95% yield).Compound 6 (125mg) is dissolved in 20mL bis- It in chloromethanes, is added EDC (130mg, 0.68mmol), NHS (115mg, 1mmol), reacts 20 hours under protection of argon gas.Reaction Liquid is washed 3 times with 20mM sodium bicarbonate solution 30mL, is washed one time with saturated brine 30mL, is spin-dried for after organic phase is dry, is obtained white Solid chemical compound 7 (IBT-114,142mg, 80% yield).NMR(400MHz,CD3OD):3.84(m,1H),3.50(m,1H), 3.29(s,2H),2.79(m,1H)2.72(s,4H),2.45(m,1H),2.50(m,4H),1.52(m,4H),0.85(t,6H)。 ESI-MS (M+H)+(calculating) 333.20, (actual measurement) 333.17.

The synthesis of embodiment 2:T-119, synthetic route are as follows:

The synthesis of T-119, asterisk represents 13C or 15N on corresponding position in synthetic route.

Compound 8 (Nosyl-Gly-OH, 15N, 2-13C, 524mg, 2mmol) is dissolved in 10mL DMF, and solid is added Sodium carbonate (636mg, 6mmol) and iodopropane (590 μ L, 1.02g, 6mmol) stir 1 hour, filter white solid, and rotation is steamed Hair removes major part DMF, and methylene chloride 30mL is added, washs 3 with 20mM sodium bicarbonate aqueous solution and saturated brine 50mL respectively Time, it removes methylene chloride and obtains 654mg white compound 9 (95% yield).Compound 9 (654mg) is dissolved in the KOH/ of 20mL In MeOH saturated solution, 400 μ L 2 mercapto ethanols are added, reaction is stirred 12 hours under nitrogen protection.Rotary evaporation removes first Alcohol.Solid residue is directly dissolved in 10mL water, is adjusted to pH~10 or so with dilute hydrochloric acid, then, addition sodium hydroxide (80mg, 2mmol), dissolution Boc2O (525mg, 2.4mmol) is slowly added dropwise to the aqueous solution of compound 10, reaction 2 in 10mL acetone Hour.Rotary evaporation remove acetone, with dilute hydrochloric acid adjust pH~2, ethyl acetate 20mL extract 3 times, it is organic mix after be evaporated Obtain white compound 11 (350mg, 1.62mmol, 85% yield).

Compound 1 (β-Ala-OBzl.HCl, 215mg, 1mmol) and (Nosyl-Gly (the Pr)-OH, 15N, 2- of compound 11 13C, 220mg, 1mmol) it is dissolved in 20mL methylene chloride, it is added EDC.HCl (229mg, 1.2mmol), reaction is protected in argon gas Shield is lower to be carried out 20 hours, and reaction solution is washed three times with 25mM dilute hydrochloric acid 30mL, is removed, is obtained with Rotary Evaporators after organic phase is dry 298mg oily compound 12 (0.8mmol, 80% yield).The EtOAc solution of compound 12 (298mg) addition 50mL HCl (1M) is stirred 2 hours, and the white precipitate of appearance is collected by filtration to obtain 230mg compound as white solid 13 (0.74mmol, 92% production Rate).Compound 13 (230mg) is dissolved in 20mL methanol, is added propionic aldehyde (13C3,80 μ L, 65mg), and 45mg cyano is then added Sodium borohydride reacts 2 hours.Methanol is removed with Rotary Evaporators, crude product methylene chloride: ethyl acetate (9:1) is in silicagel column Upper purification obtains oily compound 5 (160mg, 0.52mmol, 70% yield).Compound 5 is dissolved in 20mL methanol, is added After 20mg Pd/C, a hour is reacted under hydrogen balloon effect.Remove solvent obtain compound as white solid 6 (115mg, 0.50mmol, 96% yield).Compound 6 (115mg) is dissolved in 20mL methylene chloride, addition EDC (130mg, 0.68mmol), NHS (115mg, 1mmol) reacts 20 hours under protection of argon gas.Reaction solution 20mM sodium bicarbonate solution 30mL is washed 3 times, is washed one time with saturated brine 30mL, is spin-dried for after organic phase is dry, obtain compound as white solid 7 (IBT-119, 120mg, 0.36mmol, 72% yield).NMR(400MHz,CD3OD):3.66(m,2H),3.41(d,1H),3.17(d,1H), 2.72(s,4H),2.61(m,3H),2.50(m,2H),2.38(m,1H),1.64(m,1H),1.52(m,2H),1.40(m,1H), 0.97(m,1.5H),0.85(t,3H),0.73(m,1.5H).ESI-MS (M+H)+(calculating) 333.20, (actual measurement) 333.35.

The invention also discloses a kind of IBT-10PLEX reagents to be used for polypeptide quantitative analysis, and key step includes label, point Sub- fragmentation, quantitative three steps.After most 10 polypeptide samples can be marked respectively by IBT-10PLEX reagent, mix, Then it is separated on liquid chromatogram LC, and then generates fragments characteristic peak and reagent reporter group in MS/MS, according to characteristic peak Qualitative, quantitative analysis is carried out to polypeptide with the intensity of reporter group.

Wherein, polypeptide is prepared by the following:

(1) from the albumen of cell extraction

(2) pass through trypsin digestion

(3) desalination obtains mixtures of polypeptides

Wherein polypeptide is obtained from cell culture or other samples.

Specific quantitative analysis method 3, embodiment 4, embodiment 5, embodiment 6 as the following examples.

The optimization of embodiment 3:IBT-10PLEX label reaction condition

The optimization of Fig. 1: IBT-10PLEX label reaction condition.(A) ratio of IBT-10PLEX reagent and peptide imitates label The influence of rate.(B) influence of the label reaction time to labeling effciency.

As shown in Figure 1, the significant notation of polypeptide is the prerequisite with isobar reagent accurate quantitative analysis albumen.In general, It can carry out the mark rate of representative peptide relative to the total quantity of the peptide of identification with the label peptide quantity of identification.For system optimization IBT The condition of label mainly includes following several respects: 1) for dissolving the buffer TEAB (triethyl ammonium bicarbonate) of mixtures of polypeptides Concentration, 2) ratio between labelled reagent and polypeptide sample, 3) the label time.Optimal label reaction condition is obtained by contrast Are as follows: 1) the TEAB solution concentration for dissolving polypeptide sample is 200mM TEAB solution；2) ratio of IBT-10PLEX reagent and peptide Rate is 10；3) label reaction Best Times are 2 hours.Therefore, in actual operation by IBT-10PLEX reagent be dissolved in Polypeptide mixing in the triethyl ammonium bicarbonate of 200mM, is marked reaction.Label reaction is reacted at room temperature, and 2 hours Trifluoroacetic acid (TFA) end mark process is added afterwards, and injected sample carries out separation analysis to sample into LC-MS/MS system.

Optimal LC-MS/MS mass spectral analysis condition needed for embodiment 4:IBT-10PLEX labelled protein quantitative analysis

Optimal LC-MS/MS mass spectral analysis condition needed for Fig. 2: IBT-10PLEX labelled protein quantitative analysis.(A) divide The influence that resolution distinguishes reporter group ion.(B) NCE (orthogonal collision energy) to IBT labeling polypeptide identify and IBT report from The influence of sub- intensity.

As shown in Fig. 2, LC-MS/MS mass spectral analysis condition optimal needed for IBT-10PLEX labelled protein quantitative analysis. Liquid chromatogram separates the polypeptide sample after IBT-10PLEX label, and mixed sample (can up to 10 kinds mixing samples) can be adopted With nanometer reverse phase (RP) chromatographic column (5- μm) Hypersil C18,75 μ m 150mm, Thermo Fisher Scientific) Or the chromatographic column of similar functions carries out peptide molecule separation.10 kinds of molecular label quality of IBT-10PLEX reporter group are distinguished Within the scope of 114 to 119Da, each reagent label contains the combination of the different number and location of independent 13C and/or 15N, no The Tiny Mass difference of same isotopic molecule label carries out quality difference by high resolution mass spec, and realization analyzes and identifies and determines Amount.The signal strength of signal reports ion for quantitative analysis is not less than the 5% of sample peak-peak.

In the proteomics based on label is quantitative, mass spectrographic resolution ratio, which influences, separates adjacent report ion (example Such as, 115C is to 115N) and detection more polypeptide fragment between balance.By making sample with the tryptic digest of alpha-casein To test the analysis parameter of MS2, compare and obtain with other MS2 using identical conclusion is reported, i.e., resolution ratio be 350000 string Connection mass spectrum such as uses Orbitrap Q Exactive (Thermo Fisher Scientific, Waltham, MA) or similar functions It is best that high-resolution mass spectrometer, which carries out identification and quantification analytical effect to the mixtures of polypeptides of label,.Meanwhile the standard of MS2 is hit The value for hitting energy NCE compares analysis between 24% and 36%, the results showed that NCE value can reflect when being 30% Determine to realize optimum balance between the segment of polypeptide and quantitative IBT reporter group quantity.

The accuracy of embodiment 5:IBT-10PLEX reagent quantitative

The accuracy of Fig. 3: IBT-10PLEX reagent quantitative.(A) assessment of IBT-10PLEX labeling effciency.(B)IBT- The quantitative linearity scope of 10PLEX label.(C) accuracy of the IBT-10PLEX label to the sample relative quantification of different proportion. (D) complex sample background marks quantitative influence degree to IBT-10PLEX.

As shown in figure 3,1) for the assessment of labeling effciency: being marked respectively using 10 kinds of different labels in IBT-10PLEX The Hela cell extraction albumen of equivalent crossed through trypsin digestion.By the albumen sample of the equivalent marked by different labels after label Product mixing, by LC-MS/MS identification and quantification, the results showed that, using quality be 114 molecular label label sample as control, The sample size ratio of other 9 kinds of molecular labels label is close to 1.0.The label and quantitative result of 10 kinds of reagents of IBT-10PLEX Between almost without quantitative deviation.

2) IBT-10PLEX quantifies the assessment of series range: multiple experiments confirm that the quantization series of IBT is at least up to 50 times.

First assessment experiment, the tryptic digest peptide of cow's serum (BSA) are to be tried respectively with independent IBT-10PLEX Agent label, and mixed according to certain ratio, mixed proportion is 114:115N:115:116N:116C:117N: 117C:118N:118C:119=1:3:9:25:50:1:3:9:25:50.It is as a reference point with T-116C, report the intensity of ion Linear relationship is presented within the scope of 50 times, with theoretical calculation (slope=1.10, R2=0.97).

Second assessment experiment, the tryptic digest peptide of HeLa cell protein is respectively by each IBT-10PLEX reagent mark Note, and be to mix in varing proportions: 114:115N:115C:116N:116C:117N:117C:118N:118C:119=3:5: 10:1.5:1:1:1.5:10:5:3.Using T-117N as opposite reference value, find that most of sample measurement intermediate values are close after analysis Desired value.

Third assessment experiment: the relative quantification result using Escherichia coli peptide as A in background check complex sample Influence.The tryptic peptide of BSA is mixed, 114:115N by IBT-10PLEX separate marking with fixed proportion: 115C:116N:116C:117N:117C:118N:118C:119=1:2:4:8:10:1:2:4: 8:10；In comparative experiments, mix The Escherichia coli peptide for entering equivalent is marked together with BSA, and wherein the mixed ratio of BSA remains as 1:2:4:8:10:1:2:4: 8:10.Two groups of experiments, finally total peptide amount is not equal after the sample mixing of isolabeling, and analyzes for LC-MS/MS.Do not drawing In the group for entering Escherichia coli peptide, it is 0.97 that the slope for quantifying linear dependence, which is 0.83, R2 value, and is being mixed with Escherichia coli peptide In background group, the slope of linear fit is 0.45, R2 0.96.Sample background magazine can generate label MS signal as the result is shown It influences.But nevertheless, quantization be still it is linear, respective labels MS signal system decaying, do not influence quantitative result. Therefore it is feasible that IBT carries out Relative quantification in the system with complex background.

Analysis of the embodiment 6:IBT-10PLEX reagent for phosphoeptide in HeLa cell

Analysis of Fig. 4: the IBT-10PLEX reagent for phosphoeptide in HeLa cell.(A) phosphoeptide in HeLa cell is analyzed Flow chart.(B) quantity of the phosphoeptide of identification and quantification.(C) in the quantity of the phosphoeptide of different range relative abundance variation. (D) curve that the relative abundance of representative phosphoeptide changes over time.

As shown in figure 4, analysis of the BT-10PLEX reagent for phosphoeptide in HeLa cell:

1) cell culture: the product HeLa cell bought from ATCC is maintained at provided and contains 10% fetal calf serum (FBS) in DMEM solution, 37 DEG C of temperature control, and keep 5%CO2.Cell is provided in serum free medium and contains 150ng/ The EGF solution of mL carries out stimulation culture in 1,5,10,15,20,30,40,50 and 60 minute respectively.Different EGF thorns are contacted later Cell is harvested by centrifugation in sharp groups of cells, and is washed twice with PBS to remove remaining EGF.

2) HeLa cell protein extracts, and digestion and label label: takes 300 grams to extract from different groups of HeLa cells respectively Protein, by trypsin digestion and desalination.It is marked and is reacted with 1:10 (w/w) mixing with IBT-10PLEX reagent, Enrichment extraction is carried out to phosphoeptide therein later.

3) enrichment and separation of phosphoeptide: the phosphoeptide of IBT label has the microballoon of TiO2 coating to be enriched with using surface, It is carried out by high pH reverse phase classification separating kit (20mg, Thermo Scientific, Rockford, lL).Using different It (is respectively 5,7,8,9,10,11,13,15,17,20,25 and 50% acetonitrile containing front three amine concentration that acetonitrile concentration, which gradually elutes, Solvent).It is then combined with into 6 components and is separated and identified using LC-MS/MS.

4) mass spectral analysis condition: level-one MS1 scanning range is 350 to 2000m/z, scanning resolution 70000, lowest signal Intensity is 20000.Second level MS/MS scanning resolution is that 35000. lowest signal-to-noises are set as 1.5.

5) database search: protein identification and quantitatively using iQuant software using iPeak search (drawn by three search Hold up integrated MyriMatch v2.2.10165, X！Tandem v2017.2.1.2 and MS-GF+v2017.01.13).Identification of Fusion Protein Utilize SwissProt human data library (in April, 2017 publication) and decoy sequence.Design parameter setting are as follows: the mirror of albumen and peptide Determine error rate (FDR) to be set as less than 1%；Select trypsase as enzyme-specific, each peptide at most contains a mistake cutting； Fixed modification includes IBT-10PLEX (N-term), IBT-10PLEX (K), carbamo, lmethyl (C) and variable modification packet It includes oxidation (M), deamination (N, Q), IBT-10PLEX (Y) and phosphorylation (S, T, Y)；MS1 quality error value 20ppm, MS2 segment Quality error 0.1Da；The confirmation that only those phosphate phsophoRS score value is more than 0.75 is phosphate, and it is fixed to calculate phosphoeptide The pval of amount is converted into qval value according to Cauchy distribution.

6) the Phospoprotein expression that IBT-10PLEX reagent quantitative is stimulated through EGF: phosphorylating protein group is in EGF difference Between length (respectively 1,5,10,15,20,30,40,50,60 minutes) stimulation in produce Phospoprotein expression variation, benefit The function that can mark and quantify up to 10 kinds different samples simultaneously with IBT-10PLEX reagent always identifies that albumen number is 1971 A, total phosphoeptide that identifies is 5361, wherein have a determining phosphorylation site has 4882.

Embodiment 7

Present embodiment discloses a kind of new isobar tagging reagents, isobar tagging reagents contain 13C's and 15N Equivalent dystopy isotopic tag, isobar tagging reagents include following structure A, structure B, structure C, structure D or structure E One or more of them:

The technical means disclosed in the embodiments of the present invention is not limited only to technological means disclosed in above embodiment, further includes Technical solution consisting of any combination of the above technical features.It should be pointed out that for those skilled in the art For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as Protection scope of the present invention.

Claims

1. a kind of polypeptide quantitative analysis method, it is characterised in that: use isobar tagging reagents, be marked and determine to polypeptide Amount；Isobar tagging reagents include 10 kinds of labels.

2. a kind of polypeptide quantitative analysis method as described in claim 1, it is characterised in that: isobar tagging reagents include 10 A identical label of chemical structure, the different location in each label contain 13C or 15N isotope, 10 labels are as follows: T-114、T-115N、T-115C、T-116N、T-116C、T-117N、T-118N、T-117C、T-118C、T-119。

3. a kind of polypeptide quantitative analysis method as claimed in claim 2, it is characterised in that: isobar tagging reagents include MS/MS can shear key and can be marked polypeptide and quantifying containing isotopic tag with the group of polypeptides reactive；It is described The structure of each label of isobar tagging reagents includes following formula: reporter group-balance group-activated group, wherein reporting Group and balance group, which pass through MS/MS, can shear key connection, and activated group reacts to form stable amido bond with the amino of polypeptide； Reporter group+balance group quality in each label is identical；Concrete structure formula containing isobar tagging reagents It is as follows:

T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N, T-117C, T-118C, T-119 structural formula As follows, wherein asterisk indicates that this position is corresponding 13C or 15N:

。

4. a kind of polypeptide quantitative analysis method as claimed in claim 3, it is characterised in that: reporter group and balance group pass through MS/MS can shear key connection, and for the quality of reporter group in the range of 114 to 119Da, reporter group contains 10 kinds of differences respectively Isotope 13C and 15N mass combination, balance group contains the isotope of corresponding 13C and 15N, reporter group and balance The quality summation of group is completely the same in each reagent.

5. a kind of polypeptide quantitative analysis method as described in claim 1-4, it is characterised in that: polypeptide quantitative analysis includes following Step: label, quantifies mass spectrometric fragmentation, polypeptide is marked using isobar tagging reagents, the polypeptide after label exists Fragments characteristic peak is generated in MS/MS, and polypeptide is quantified according to the intensity of characteristic peak；

The polypeptide is prepared by the following:

(1) from the albumen of cell extraction

(2) pass through trypsin digestion

(3) desalination obtains mixtures of polypeptides.

6. a kind of isobar tagging reagents, it is characterised in that: isobar tagging reagents contain 10 kinds of equivalent of 13C and 15N Dystopy isotopic tag, 10 labels are as follows: T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N, T- 117C,T-118C,T-119；Each equivalent dystopy isotopic tag includes 13C and/or 15N isotope atom；Isobar mark Signing agent structure includes following formula: reporter group-balance group-activated group；Reporter group and balance group pass through can shear key Connection, shear key are preferentially broken in MS/MS, are generated reporter group ion, are respectively provided with different molecular formula and quality, are reported Specific molecular formula and the accurate mass for accusing group ion are as follows:

T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-117C, T-118N, T-118C, T-119 structural formula As follows, wherein asterisk indicates that this position is corresponding 13C or 15N:

。

7. a kind of isobar tagging reagents as claimed in claim 7, it is characterised in that: the wherein quality of the reporter group In the range of 114 to 119Da, the reagent contains the same position of the different number and location independently selected from 13C and/or 15N Plain atom combination, and identification and analysis is carried out by mass spectrum；Steps are as follows for identification and analysis:

8. a kind of method for being synthetically prepared any one isobar tagging reagents of claim 6-7, it is characterised in that: use and contain There are the raw material β-Ala-OBzl of different isotopes, Boc-Gly-OH, propionic aldehyde synthesizes 7 kinds of isobar labels according to following methods T-114,T-115N,T-115C,T-116N,T-116C,T-117N,T-118N；

Specific step is as follows:

2) compound 3 sloughs Boc group in acid condition, forms compound 4；

Process route is as follows:

。

9. a kind of method for being synthetically prepared any one isobar tagging reagents of claim 6-7, it is characterised in that:

Using the raw material β-Ala-Obzl containing different isotopes, Boc-Gly-OH, propionic aldehyde, according to following methods synthesize 3 kinds it is same Measure dystopy tagging reagents, including T-117C, T-118C, T-119；Specific step is as follows:

1) 2-Nosyl-Gly-OH (compound 8) reacts to form compound 9 with iodopropane；

4) β-Ala-OBzl.HCl (compound 1) and compound 11 are coupled to compound 12；

5) compound 12 sloughs Boc group in acid condition, forms compound 13；

6) compound 13 and propionic aldehyde are in reducing agent (NaCNBH₃) compound 5 is formed under effect；

Process route is as follows:

。

10. a kind of isobar tagging reagents, it is characterised in that: isobar tagging reagents contain the equivalent dystopy of 13C and 15N Isotopic tag, the isobar tagging reagents include following structure A, structure B, structure C, structure D or structure E wherein It is one or more:

。