CN116337567A - Quick and efficient chemical proteomics sample preparation method - Google Patents

Abstract

The application discloses a quick and efficient preparation method of chemical proteomics samples, relates to the field of proteomics, designs and synthesizes agarose microspheres with surfaces coated with cleavable linker compounds, wherein free ends of the cleavable linker compounds contain azide groups, and has better compatibility with the existing alkynyl probes. The probe-labeled peptide Duan Te can be captured on the surface of the agarose microsphere in a specific manner through a one-step high-efficiency click chemical reaction, and due to covalent connection between the agarose microsphere and the peptide fragment, a very strong denaturant or surfactant can be used for washing impurities of a medium so as to remove nonspecific adsorption as much as possible, and finally the probe-labeled peptide fragment can be dissociated from the agarose microsphere in a high-efficiency manner through a cleavage treatment. The sample preparation method simplifies the complicated click chemistry and streptavidin enrichment into one-step rapid click chemistry reaction, and greatly simplifies the operation flow.

Description

Quick and efficient chemical proteomics sample preparation method

Technical Field

The invention relates to the field of proteomics, in particular to a rapid and efficient chemical proteomics sample preparation method.

Background

Activity-based protein analysis (Activity-based Protein Profiling, ABPP ¹ ) The technology provides an efficient analysis platform for global characterization of protein functions. In the ABPP technical system, different chemical probes can specifically capture the active center of enzyme, functional sites of protein, post-translational modification and the like, and the detection of probe labeled protein is realized by combining affinity enrichment and mass spectrometry.

Chemical probes typically comprise a "reactive group" that reacts with a functional site of a protein and a "reporter group" that can be used for enrichment or detection. Where "reporter groups" may also be replaced with bioorthogonal groups (e.g., alkynyl groups) and then attached to the label that can be enriched by click chemistry. The probe tag peptide Duan Te can be cleaved from the affinity medium by introducing a cleavable linker (e.g.the most classical TEV cleavage linker) into the enrichment tag, and this strategy derived from ABPP is named active protein assay technique based on tandem orthogonal protease hydrolysis (Tandem Orthogonal Proteolysis Activity-based Protein Profiling, TOP-ABPP ² )。

In subsequent studies, the application scope of TOP-ABPP was further expanded, mainly in three ways: 1, introducing an isotope labeling strategy to realize specific quantification of probe labeled peptide fragments among different groups; 2, developing different types of cleavable linker for improving the dissociation efficiency of the probe labeled peptide fragment; 3, developing different chemical reaction groups to capture different reactive residues, enzyme active centers and posttranslational modifications. To date, TOP-ABPP has been widely used in the fields of drug target spot discovery, lead compound screening, etc.

However, the conventional TOP-ABPP sample preparation procedure is long, and it usually takes 2 to 3 days or more to prepare a batch of samples, as shown in FIG. 1 (b). Complex operation procedures introduce a large number of variables, which affect quantitative reproducibility between samples and limit throughput of sample preparation. Meanwhile, the enrichment tag with the cleavable linker and the commercial streptavidin enrichment medium are expensive, so that the experimental cost is high. To solve the above problems, the applicant wishes to develop a TOP-ABPP sample preparation procedure that is more simplified, saving time and reagent consumption costs.

The references are as follows:

1.Cravatt,B.F.；Wright,A.T.；Kozarich,J.W.,Activity-based protein profiling:from enzyme chemistry to proteomic chemistry.Annual review of biochemistry 2008,77,383-414.

2.Weerapana,E.；Speers,A.E.；Cravatt,B.F.,Tandem orthogonal proteolysis-activity-based protein profiling(TOP-ABPP)--a general method for mapping sites of probe modification in proteomes.Nature protocols 2007,2(6),1414-25.

3.Li,Z.；Liu,K.；Xu,P.；Yang,J.,Benchmarking Cleavable Biotin Tags for Peptide-Centric Chemoproteomics.Journal of proteome research 2022,21(5),1349-1358.

4.Yang,J.；Gupta,V.；Tallman,K.A.；Porter,N.A.；Carroll,K.S.；Liebler,D.C.,Global,in situ,site-specific analysis of protein S-sulfenylation.Nature protocols 2015,10(7),1022-1037.

5.Kuljanin,M.；Mitchell,D.C.；Schweppe,D.；Gikandi,A.S.；Nusinow,D.；Bulloch,N.J.；Vinogradova,E.V.；Wilson,D.；Kool,E.T.；Mancias,J.D.；Cravatt,B.F.；Gygi,S.,Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries.Nature biotechnology 2021,39(5),630-641.

6.Hurben,A.；Erber,L.N.；Tretyakova,N.；Doran,T.,Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized Catecholamine.ACS chemical biology 2021,16(11),2581-2594.

7.Weerapana,E.；Wang,C.；Simon,G.M.；Richter,F.；Khare,S.；Dillon,M.B.；Bachovchin,D.A.；Mowen,K.；Baker,D.；Cravatt,B.F.,Quantitative reactivity profiling predicts functional cysteines in proteomes.Nature 2010,468(7325),790-5.

disclosure of Invention

The invention provides a chemical proteomics sample preparation method, which solves the problems of longer preparation flow, complicated method, higher cost and the like of the traditional TOP-ABPP sample.

In order to solve the problems, the invention provides the following technical scheme:

a rapid and efficient method for preparing a chemical proteomic sample, comprising the steps of:

s1, adding an alkynyl probe into whole cell lysate for marking, adding methanol chloroform to precipitate protein into the marked whole cell lysate, and centrifuging to discard supernatant;

s2, washing the protein precipitate with precooled methanol, re-suspending the protein precipitate with a phosphate buffer solution containing SDS, and adding dithiothreitol for reaction to obtain a mixed solution I;

s3, adding iodoacetamide into the first mixed solution for light-shielding reaction to obtain a second mixed solution;

s4, adding methanol and chloroform into the mixed solution II for precipitation, washing the precipitate with precooled methanol, and then re-suspending the precipitate with PBS to obtain alkynyl probe marked protein liquid;

s5, adding enrichment microbeads into the alkynyl probe marked protein liquid, and fixing protein or peptide fragments marked by the alkynyl probe on the surface of the enrichment microbeads through CuAAC; the enrichment microbeads are microbeads coated with cleavable compounds, wherein the cleavable compounds comprise cleavable groups, one end of each cleavable group is connected with an amino group, the other end of each cleavable group is connected with an azide group, and the cleavable compounds are combined on the surfaces of the enrichment microbeads through amino coating, as shown in a graph (a) in fig. 1; the azide group and the alkynyl in the alkynyl probe undergo click chemical reaction;

s6, centrifuging after the click chemistry reaction is finished, discarding the supernatant, taking the enrichment microbeads, washing the enrichment microbeads with urea aqueous solution, PBS and water in sequence, cutting off cleavable groups by utilizing a cutting reaction after washing, releasing protein or peptide fragments from the enrichment microbeads in a free manner, centrifuging after the cutting reaction is finished, taking the supernatant, and spin-drying the supernatant in vacuum to obtain the proteomics sample.

Preferably, the cleavage reaction is an acid cleavage reaction or a photo cleavage reaction, wherein the acid cleavage reaction is a reaction in which enriched microbeads with labeled proteins or peptide fragments bound on the surface are resuspended in an acid solution; the acid is at least one of formic acid, acetic acid, propionic acid or hydrochloric acid;

the photocleavage reaction is to resuspend the enriched microbeads with the marked proteins or peptide fragments bound on the surface in methanol solution, and then give light to carry out the photocleavage reaction.

Preferably, when the cleavage reaction is an acid cleavage reaction, the cleavable compound is an acid cleavage linker compound having a structure represented by formula (I):

preferably, when the cleavage reaction is a photocleavable reaction, the cleavable compound is a photocleavable linker compound having a structure represented by formula (II):

preferably, the enriched microbeads are prepared as follows:

(1) Preparing a 40mM stock solution of an acid cutting linker compound or a light cutting linker compound by using DMSO;

(2) Taking a proper amount of NHS modified agarose beads, ensuring that 50 mu L of solid medium is used for each reaction, washing the NHS modified agarose beads by precooled hydrochloric acid, re-suspending the NHS modified agarose beads in 10mL of Triton PBS solution, adding a stock solution, coating and incubating; the volume ratio of the NHS modified agarose bead heavy suspension to the stock solution is 6-10: 1, a step of;

(3) Centrifuging after incubation, discarding supernatant, and sealing with ethanolamine solution;

(4) Centrifuging, discarding the supernatant, washing with PBS, and re-suspending the solid in glycerol PBS solution to obtain the enriched microbeads.

Preferably, the pre-chilled hydrochloric acid is 1mM, the Triton PBS solution is 0.1% Triton PBS solution, and the ethanolamine solution is 0.1M; the ethanol amine solution is blocked at room temperature for 6 hours or at 4 ℃ overnight; the glycerol PBS solution is 30% glycerol PBS solution.

Preferably, the alkynyl probe in the S1 is a bio-orthogonal probe, and the final concentration of the alkynyl probe in the whole cell lysate is 1-1000 mu M;

the final concentration of dithiothreitol in the S2 is 5-15 mM, and the reaction condition of the dithiothreitol is 30-40 ℃ for 20-40 min; the content of SDS in the phosphate buffer solution is 1.0% -1.5%;

the final concentration of the iodoacetamide in the S3 is 10-30 mM, and the light-shielding reaction condition is 30-40 ℃ and the light-shielding reaction is carried out for 20-40 min;

the protein concentration of the alkynyl probe marked protein liquid in the S5 is 1-5 mug/mu L, and the CuAAC is prepared by adding 50 mu L of enrichment microbeads, 40 mu L of 25mM Cu-BTTAA stock solution and 120 mu L of 5% sodium ascorbate into each 1mL of alkynyl probe marked protein liquid, and reacting for 2 hours at 25-29 ℃;

the concentration of urea in the S6 is 4-8M.

Preferably, the alkynyl probe marked protein solution is subjected to pancreatin digestion for 4-12 hours before the step S5 is carried out, so as to obtain an alkynyl probe marked peptide solution, and the alkynyl probe marked peptide solution and the enriched microbeads are subjected to CuAAC reaction.

Preferably, 200 mu L of 2% formic acid aqueous solution is added into each 50 mu L of enrichment microbeads for reaction for 30-120 min after re-suspension;

the photo-cutting reaction is to re-suspend the enriched microbeads with 50-70% methanol aqueous solution, and light at 365nm in a light-transmitting carrier for 20-50 min.

The invention has the beneficial effects that:

the agarose microsphere with the surface coated with the cleavable linker compound is designed and synthesized, and the free end of the cleavable linker compound contains an azide group and has better compatibility with an alkynyl probe used in the conventional TOP-ABPP experiment. The probe-tagged peptide Duan Te can be captured on the agarose microsphere surface by a one-step efficient click chemistry reaction, as shown in fig. 1 (c). Because of covalent linkage between agarose microsphere and peptide segment, very strong denaturant or surfactant can be used to wash the medium to remove nonspecific adsorption as much as possible, and finally the probe-labeled peptide segment can be dissociated from agarose microsphere with high efficiency by acid or photo-cleavage treatment. The newly developed TOP-ABPP sample preparation process simplifies the complicated click chemistry and streptavidin enrichment into a one-step rapid click chemistry reaction, and greatly simplifies the operation process. Compared with the traditional TOP-ABPP, the operation time of the superTOP-ABPP can be reduced to 9 hours, and the preparation of samples can be completed in one day as shown in a graph (d) in fig. 1.

Drawings

In order to more clearly illustrate the technical solutions of the present application, the drawings that are needed in the embodiments will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a diagram comparing a conventional sample preparation method with the sample preparation method of the present application. Wherein, (a) the figure is coated with the preparation flow of the cleavable linker compound agarose microsphere; (b) FIG. is a flow chart of a conventional TOP-ABPP sample preparation; (c) FIG. is a flow chart of the preparation of a superTOP-ABPP sample according to the invention; (d) The figure shows a time-consuming comparison of conventional TOP-ABPP and superTOP-ABPP strategies.

FIG. 2 is a parallel comparison result of the identification effect of super TOP-ABPP and conventional TOP-ABPP on the modification site of the probe. Wherein, the (a) and (b) are the statistics of the number of the probe modification sites obtained by identifying TOP-ABPP sample and superTOP-ABPP sample. (c) The graph shows the signal intensity of peptide fragments in the superTOP-ABPP sample and TOP-ABPP sample compared with the result graph.

FIG. 3 shows the results of chemical proteomic analysis of the SuperTOP-ABPP method and TOP-ABPP method for microsamples, respectively. Wherein (a) the mass spectrum detection result of TOP-ABPP trace sample is shown in the figure; (b) The figure shows the mass spectrum detection result of the superTOP-ABPP micro sample; (c) The figure is a comparative statistical graph of analysis results obtained based on two sample preparation methods; (d) The figure is a comparison of the cost and expense of two sample preparation methods.

FIG. 4 is a graph showing the evaluation result of the quantitative accuracy of superTOP-ABPP. Wherein, (a) is the standard deviation of the pooled samples after enrichment; (c) The graph shows the standard deviation of the combined and then enriched samples, and the graph (b) shows the linear relationship between the detection value of the light-weight ratio and the theoretical value of the combined and enriched samples; (d) The graph shows the linear relationship between the detection value of the light-weight ratio and the theoretical value of the combined and then enriched sample.

FIG. 5 shows the results of the SuperTOP-ABPP strategy for quantitative detection of hyperreactive cysteines. (a) Overall distribution of peptide fragment intensity ratios under 100 μm and 10 μm probe labeling; (b) A correlation analysis of the number of identified highly active cysteines between three replicates and (c) quantification; (d) The known high activity cysteine sites can be effectively identified by superTOP-ABPP, and have better correlation with the results obtained by the traditional isoTOP-ABPP.

FIG. 6 is a comparison of the identification efficiency of acid cut and photo cut linker. Wherein, (a) the figure shows the mass spectrometry detection result of a sample prepared by enrichment of microbeads coupled with an acid-cutting linker compound; (b) FIG. is a graph showing the results of mass spectrometry detection of samples prepared by enrichment of microbeads coupled with a photocleavable linker compound; (c) The figure is a comparison statistical plot of the identification efficiency of two different enriched microbeads.

FIG. 7 is a CY74 compound ¹ H NMR spectrum.

FIG. 8 is a CY75 compound ¹ H NMR spectrum.

FIG. 9 shows CY62 compounds ¹ H NMR spectrum.

FIG. 10 shows CY64 compounds ¹ H NMR spectrum.

FIG. 11 is a CY66 compound ¹ H NMR spectrum.

FIG. 12 is a comparison of conventional TOP-ABPP with SuperTOP-ABPP identification using enriched microbeads for different probe-tagged proteins; wherein, (a) is a lysine-reactive STPyne probe and a labeling identification result thereof. (b) The figure shows the identification result of cysteine-reactive iodoacetamidate alkynyl probe IAyne and its mark. In the figure azide beads represent SuperTOP-ABPP samples prepared using enrichment microbeads.

FIG. 13 is data for MGOyne target identification using superTOP-ABPP data. (a) The figure is a flow chart of the whole experimental operation, and MGO and MGOyne are used for competing to screen the sites with the marked weakening of MGOyne under the MGO treatment condition as MGO sensitive sites; (b) The figure shows the distribution of the coefficient of variation of the quantified peptide fragments between the replicates; (c) The graph shows the signal intensity ratio distribution of peptide fragments under +/-MGO treatment conditions; (d) The graph shows the correlation of the ratio of the signal intensities of the peptide fragments between the two replicates.

Detailed Description

The following describes specific embodiments of the present application in detail. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.

Example 1: synthesis of acid cut Linker Compound CY75

Reagents and conditions in the synthesis reaction: (a) NaN (NaN) ₃ 0-65 ℃, overnight, 90%; (b) Ph (Ph) ₂ SiCl ₂ TEA, 0-rt, 36h,47.5%; (c) 20% piperidine, 0-rt, 21%.

Synthesis of Compound CY72

6-bromo-1-hexanol (0.96 mL,6 mmol) was dissolved in a solution of super-dry DMF (15 mL), ice-bath, and cooled to 0deg.C. Sodium azide (0.96 g,12mmol,2.0 equiv.) was added slowly. The mixture was slowly heated to 65 ℃ and stirred overnight. After cooling to room temperature, the mixture was filtered, saturated brine (30 mL) was added, and the mixture was extracted with ethyl acetate (20 mL. Times.3). The organic phases were combined, anhydrous Na ₂ SO ₄ Drying, filtration and vacuum distillation gave the crude product.

Synthesis of Compound CY74

Ph under ice bath ₂ SiCl ₂ (630. Mu.L, 3.0 mmol) and Et ₃ N (832. Mu.L, 6.0 mmol) was added to 15mL of methylene chloride. Compound 72 (143 m) was slowly added dropwiseg,1.0 mmol) in dichloromethane (1.5 mL) and then slowly bring the reaction to room temperature. After 12 hours, 73 (2034 mg,6.0 mmol) was added and stirring was continued for 24 hours. Dichloromethane extraction (20 ml×3), combining the organic phases, back-extracting the organic phases with saturated brine, anhydrous Na ₂ SO ₄ Drying, and vacuum concentrating in water bath below 30deg.C. Column chromatography purification (PE: ea=10:1 to 5:1) gave 74 (315 mg, 47.5%) as a white solid as shown in figure 7, ¹ H NMR(400MHz,Chloroform-d)δ7.80–7.53(m,8H),7.46–7.26(m,10H),4.73(s,1H),4.40(d,J＝6.8Hz,2H),4.21(t,J＝6.7Hz,1H),3.77(t,J＝6.4Hz,3H),3.23–3.15(m,4H),1.68–1.24(m,17H). ¹³ C NMR(101MHz,Chloroform-d)δ156.41,144.04,141.34,134.90,133.08,130.23,127.83,127.66,127.03,125.03,119.97,66.48,62.95,60.42,51.41,47.34,41.05,32.36,32.28,29.98,28.82,26.46,25.50,25.38 21.07,14.22.HRMS(ESI):Calcd for C ₃₉ H ₄₇ N ₄ O ₄ Si[M+H] ⁺ 663.33611,found 663.33652.

synthesis of Compound CY75

74 (100 mg,0.15 mmol) was added to 10mL of dichloromethane under ice bath, and 20% piperidine (152. Mu.L) was added dropwise. After stirring for 30 minutes, the reaction mixture was warmed to room temperature. After completion of TLC detection, the reaction was extracted with dichloromethane (20 mL. Times.3), the organic layers were combined, washed twice with saturated brine, and dried over Na ₂ SO ₄ And (5) drying. Filtering, vacuum concentrating the solvent in 30deg.C water bath, and purifying by preparative thin layer chromatography (CH ₂ Cl ₂ Meoh=20:1) to give 75 (14 mg, 21%) as a pale yellow solid. As shown in figure 8 of the drawings, ¹ H NMR(400MHz,Methanol-d ₄ )δ7.67–7.57(m,4H),7.47–7.31(m,6H),3.79(q,J＝6.3Hz,2H),3.62–3.53(m,1H),3.25(t,J＝6.8Hz,1H),2.93–2.85(m,1H),1.69–1.51(m,6H),1.47–1.22(m,12H),0.90(t,J＝6.8Hz,1H). ¹³ C NMR(101MHz,Methanol-d ₄ )δ134.50,132.86,130.02,127.51,62.60,62.54,50.99,39.32,31.92,31.87,28.46,27.17,26.06,25.79,25.09,25.05.HRMS(ESI):Calcd for C ₂₄ H ₃₆ N ₄ O ₂ Si[M+H] ⁺ 441.26803,found 441.26795.

example 2: synthesis of photo-cutting linker compound GZH66

Reagents and conditions: (a) NaN (NaN) ₃ 0-90 ℃, overnight, 55.9%; (b) i) DCC, et ₃ N, NHS; ii) Boc-ethylenediamine, 33.9%; (c) 62, DSC, et ₃ N；(d)TEA,0℃,6h,25.9％.

Synthesis of Compound 62

3-Bromopropylamine hydrobromide (8.4 g,38.7 mmol) was dissolved in water, ice-cooled to 0deg.C. Sodium azide (8.5 g,130.8mmol,3.4 eq) was added slowly. The mixture was slowly heated to 90 ℃ and stirred overnight. After cooling to room temperature, the mixture was filtered, saturated brine (30 mL) was added, and the mixture was extracted with ethyl acetate (50 mL. Times.3). The organic phases were combined, anhydrous Na ₂ SO ₄ Drying, filtration and vacuum distillation gave the crude product. As shown in the figure 9 of the drawings, ¹ H NMR(400MHz,CDCl ₃ )δ3.38(t,J＝6.7Hz,2H),2.81(t,J＝6.8Hz,2H),1.73(p,J＝6.8Hz,2H),1.18(s,2H).

synthesis of Compound 64

i) 63 (1.0 g,3.3 mmol) was added to 25mL of methylene chloride under ice bath, NHS (760.0 mg,6.6 mmol) and Et were added ₃ N (1.0 g,10.0 mmol). DCC (825.2 mg,4.0 mmol) was added and the mixture was slowly brought to room temperature and stirred overnight. And (5) filtering.

ii) the filtrate obtained in i) was taken, boc-ethylenediamine (1.1 g,6.6 mmol) was added and stirred at room temperature overnight. Washing with saturated saline solution for three times, purifying the obtained product by column chromatography, and eluting with eluent with polarity of CH ₂ Cl ₂ : meoh=40: 1. spin-drying gives 500mg of pale yellow product. As shown in figure 10 of the drawings, ¹ H NMR(400MHz,CDCl ₃ )δ7.55(s,1H),7.32(s,1H),5.54(q,J＝6.2Hz,1H),4.10(t,J＝5.6Hz,2H),3.97(s,3H),3.34(m,2H),3.27–3.13(m,2H),2.41(t,J＝7.2Hz,2H),2.17(m,3H),1.54(d,J＝6.2Hz,3H),1.42(s,9H).

synthesis of Compound 65

DSC (89.6 mg,0.35 mmol) and Et were run in an ice bath ₃ N (70.8 mg,0.70 mmol) was added to 10mL DMF and 64 (100.0 mg,0.24 mmol) was added slowly and stirred overnight. 62 (70.1 mg,0.70 mmol) was added and stirred overnight. Spin-drying, warpPreparation of thin layer chromatography purification (CH ₂ Cl ₂ Meoh=20:1) to afford 65 crude product, which was directly taken to the next step.

Synthesis of Compound 66

Under ice bath conditions, the 65 crude product was dissolved in 10mL CH ₂ Cl ₂ Is a kind of medium. 1mL of trifluoroacetic acid was added and stirred for 6 hours. Preparation of thin layer chromatography purification (CH ₂ Cl ₂ : meoh=5:1). As shown in the figure 11 of the drawings, ¹ H NMR(400MHz,MeOD)δ7.61(s,1H),7.16(s,1H),6.26(q,J＝5.7Hz,1H),4.10(q,J＝3.4Hz,2H),3.95(s,3H),3.83(t,J＝5.7Hz,1H),3.55(t,J＝5.7Hz,1H),3.47(t,J＝5.8Hz,1H),3.13(t,J＝6.5Hz,2H),3.07(t,J＝5.5Hz,1H),2.63(s,1H),2.50–2.40(m,4H),2.15–2.07(m,2H),1.69(dd,J＝13.2,6.6Hz,2H),1.58(d,J＝6.3Hz,3H).

example 3: agarose microbead coupling cleavable linker compounds to prepare enriched microbeads, comprising the specific steps of:

1. CY75 synthesized in example 1 or GZH66 synthesized in example 2 were formulated with DMSO as 40mM stock solutions. GZH66 is protected from light during the whole coating process.

2. An appropriate amount of NHS modified agarose beads, hereinafter abbreviated as NHS beads (cytova, cat. No. 17037137), was taken to ensure that 50. Mu.L of solid medium was used for each reaction, after 3 times of NHS beads with 1mM ice-cold hydrochloric acid, the NHS beads were resuspended in 10mL of 0.1% Triton PBS solution, 250. Mu.L of CY75 or GZH stock solution was added for each 2mL of NHS modified agarose bead suspension, and incubated at room temperature for 6 hours or overnight at 4 ℃. The volume ratio of the NHS modified agarose bead heavy suspension to the stock solution can be between 6 and 10 according to the reaction requirement: 1.

3. The supernatant was removed by centrifugation, excess NHS groups were blocked with 0.1M ethanolamine solution, and the reaction was performed for 6 hours at room temperature or overnight at 4℃to give agarose beads coated with CY75 or GZH.

4. Centrifuging, discarding supernatant to obtain enriched microbeads, washing with PBS for 5 times, re-suspending the enriched microbeads in 30% glycerol PBS solution, packaging, and freezing in a refrigerator at-20deg.C for use.

Example 4: the TOP-ABPP method is adopted to prepare a sample, which is specifically as follows:

1. the whole cell lysate was labeled with a probe and then attached to an acid-cleavable biotin tag via CuAAC for a reaction time of 1 hour.

2. The precipitated proteins were washed 3 times with cold methanol and resuspended in 1.2% SDS PBS.

3. SDS was diluted to a concentration of 0.2% or less, and then concentrated with 100. Mu.L of streptavidin agarose beads, hereinafter referred to as beads (Thermo Scientific, cat. 20353), and reacted at 29℃for 4-6 hours.

4. The incubated beads were washed 3 times with 5mL of PBS and 3 times with 5mL of water before transferring to the screw tube.

5. The supernatant was centrifuged off, the beads were resuspended in 500. Mu.L of 6M urea, and 10mM Dithiothreitol (DTT) was added for 30min at 37 ℃.

6. 20mM Iodoacetamide (IAA) was added and reacted at 35℃for 30min in the absence of light.

7. The supernatant was removed by centrifugation, the beads were resuspended in 200. Mu.L PBS, and pancreatin was added for overnight cleavage for a reaction time of 12-16 hours.

The supernatant was centrifuged off, the beads were washed 2 times with 1mL of PBS, the beads were washed 5 times with 1mL of water, and then the probe-tagged peptide fragments were dissociated from the beads with formic acid solution.

Example 5: the preparation method of the cysteine targeting probe labeled peptide fragment sample by using the super TOP-ABPP method comprises the following steps of:

s1, adding iodoacetamide alkynyl probe IA-alkyne into whole cell lysate for marking, adding methanol chloroform to precipitate protein into the marked whole cell lysate, centrifuging, discarding, and removing redundant probes;

s2, washing the protein precipitate with precooled methanol for 2 times, re-suspending the protein precipitate with phosphate buffer solution containing 1.2% SDS, and adding 10mM Dithiothreitol (DTT) for reaction at 37 ℃ for 30min to obtain a mixed solution I;

s3, adding Iodoacetamide (IAA) with the final concentration of 20mM into the first mixed solution, and carrying out light-shielding reaction for 30min at 35 ℃ to obtain a second mixed solution;

s4, adding methanol and chloroform into the mixed solution II for precipitation, washing the precipitate with precooled methanol for 3 times, and then carrying out PBS ultrasonic resuspension precipitation, and carrying out pancreatin digestion for 4-12 hours to obtain alkynyl probe labeled peptide fragment solution;

s5, adding enrichment microbeads into alkynyl probe marked peptide fragment liquid with the concentration of 2 mug/mug, and carrying out click chemistry reaction CuAAC (copper catalyzed azide and alkynyl cycloaddition reaction), wherein the CuAAC is prepared by adding 50 mug enrichment microbeads, 40 mug 25mM Cu-BTTAA stock solution and 120 mug 5% sodium ascorbate into 1mL alkynyl probe marked protein liquid, and reacting for 2 hours at 25-29 ℃;

s6, centrifuging to obtain enrichment microbeads after the reaction is finished, washing the enrichment microbeads with 8M urea once, washing the enrichment microbeads with 1mL PBS for 2 times, washing the enrichment microbeads with 1mL water for 5 times, adding 200 mu L of 2% formic acid aqueous solution into every 50 mu L of enrichment microbeads after washing, carrying out reaction for 60min after resuspension, dissociating the probe labeled peptide from the enrichment microbeads, and centrifuging to obtain a supernatant after the reaction is finished to obtain a proteomics sample.

The method can be used for enriching probe-labeled peptide fragments after pancreatin cleavage, and can also be used for enriching probe-labeled proteins, and the pancreatin cleavage operation is not performed when the probe-labeled proteins are enriched.

Applicants have compared in parallel the identification of TOP-ABPP and SuperTOP-ABPP for probe modification sites. The applicant labeled the proteome with iodoacetamidate alkynyl probe IA-alkyne, which specifically reacted with cysteine, and then treated the samples with the procedure of superTOP-ABPP and conventional ABPP, respectively. The mass spectrum identification result shows that: under the same initial amount, TOP-ABPP identified 6953 number of modification sites and SuperTOP-ABPP identified 12814 number of probe modification sites (FIGS. 2a, b). Meanwhile, the applicant found that the median of the signal intensity Log2 of the peptide fragments identified by TOP-ABPP was about 21.7, and that of the peptide fragments in the superTOP-ABPP group was 24.1. That is, the enrichment efficiency of SuperTOP-ABPP for probe-modified peptides is about 5 times that of TOP-ABPP.

Given the higher enrichment efficiency of superTOP-ABPP, applicants hypothesize that this strategy can be used for chemical proteomic analysis of fewer samples. In a conventional TOP-ABPP experiment, the initial amount of each sample was 2mg. Applicants used herein 1/10 of the initial sample size (200 μg) of conventional TOP-ABPP to test the enrichment sensitivity of SuperTOP-ABPP. 200 μg of IA-alkyne probe-labeled whole proteomes were treated using the procedures of TOP-ABPP and SuperTOP-ABPP, respectively. Results from mass spectrometry (see fig. 3): the TOP-ABPP method identified 4604 probe-tagged peptides and the SuperTOP-ABPP method identified 7913 probe-tagged peptides, as shown in FIGS. 3 (a) - (c). The excellent peptide fragment identification performance showed that: the superTOP-ABPP strategy has great application potential in chemical proteomics analysis of micro-samples. Meanwhile, the applicant compares the experimental cost of superTOP-ABPP with that of the conventional TOP-ABPP: it takes about 61 yuan for preparing one TOP-ABPP sample, while one superTOP-ABPP sample only takes 8 yuan, which is only 13% of the cost of the conventional TOP-ABPP experiment, as shown in FIG. 3 (d) and Table 1. In summary, superTOP-ABPP is a faster, more efficient and economical chemical proteomic sample preparation procedure than traditional sample preparation methods.

Table 1. Detailed comparisons of TOP-ABPP and SuperTOP-ABPP in experimental time, reagent costs and number of identified polypeptides.

Comparison with TOP-ABPP

The applicant then evaluated the accuracy of the superTOP-ABPP strategy for quantification of probe-tagged peptide fragments and dynamic range. In current quantitative proteomics studies, amino acid stable isotope labeling of cells (stable isotope labelingby amino acids in cell culture, SILAC) is a quantitative gold standard. Light (lysine +0, arginine +0) and heavy (lysine +8, arginine +10) target MCF-7 cells were obtained using the SILAC labeling method herein. Labeling cells after lysis by using an IA-alkyne probe, and then enriching by using a strategy invented by the applicant, wherein the proportions of a light label and a heavy label are as follows: 5:1, 2:1, 1:1, 1:2 and 1:5. After enrichment, different groups of light and heavy standard samples are mixed and used as experimental groups for testing interference generated by enrichment on quantification. Simultaneously setting a group of light and heavy standard simultaneous enrichment groups after pre-mixing as a control group, wherein the control group can represent the true of the light and heavy standard peptide fragments in the sampleThe real proportion. Analysis of the mass spectrum showed that: the actual detected light-to-heavy ratio in the control group had a better linear relationship with the expected ratio (R ² =0.99) (panel b in fig. 4). The light-to-heavy ratio detected by the experimental group also has good linearity with the expected value (R ² =1.00) (d plot in fig. 4). Meanwhile, the standard deviation of the samples in the experimental group and the control group is relatively similar (a graph and c graph in fig. 4). The above results indicate that the superTOP-ABPP method has similar quantitative accuracy and linear dynamic range to that of SILAC.

For quantitative analysis of chemical proteomics, another technique most widely used today is isotope-labeled based isoTOP-ABPP ⁷ (isotopic Tandem Orthogonal Proteolysis Activity Based Protein Profiling) techniques. The prior art (Weerapana, e.; wang, c.; simon, g.m.; richter, f.; khare, s.; dillon, m.b.; bachovcin, d.a.; mowen, k.; baker, d.; cravit, b.f.; quantitative reactivity profiling predicts functional cysteines in proteins. Nature 2010, 468 (7325), 790-5.) uses isoTOP-ABPP techniques to quantitatively compare differences in cysteine signals at high and low concentrations of probe tags in the proteome, systematically analyzing high activity cysteines in cells, providing a powerful tool for the study of cysteine function. The principle is as follows: highly reactive cysteines will rapidly achieve saturation labelling at low probe concentrations. Thus, if applicants use high and low concentrations of probes to label intracellular cysteines, the ratio of signal intensities of the high-activity cysteines between the two groups should be close to1 (R: [ low:)]And 1). In this study, applicants used the method of superTOP-ABPP binding reduction dimethyl quantification to quantitatively analyze the cysteine group under high and low concentration probe labeling, while comparing with classical isoTOP-ABPP strategy for further testing the quantitative accuracy and dynamic range of superTOP-ABPP in highly complex samples. Based on the results of mass spectrometry detection: isoTOP-ABPP was quantified to 1507 probe-modified peptides in three replicates, and the superTOP-ABPP group was quantified to 1841 probe-modified peptides (panel a in FIG. 5). Wherein R is high]Low: (Low)]The peptide close to1 occupies only a small part of the total quantitative peptideIn part, this is as expected (fig. 5 b). Finally, the superTOP-ABPP strategy co-quantifies 68 of the highly reactive cysteine sites in three replicates, and the quantitative ratio of these 68 sites shows higher agreement between the three replicates (R ² =0.87 to 0.93) (fig. 5 c). The above results demonstrate the high reproducibility of the superTOP-ABPP strategy in quantification. Of these 68 sites, a range of highly reactive cysteine sites are well known to those skilled in the art, such as CKB (C283), PRMT1 (C101, ACAT1 (C126) and GSTO1 (C32). For these known highly reactive cysteine sites, the quantitative ratios of isoTOP-ABPP and superTOP-ABPP are relatively close (d-plot in FIG. 5), again confirming the quantitative accuracy of the superTOP-ABPP strategy in complex samples.

In addition to Acid Cleavable (AC) linker compounds, applicants synthesized Photocleavable (PC) GZH66, which can be coupled to NHS beads by the same scheme for enrichment of alkynyl probe-labeled peptide fragments and proteins. The specific procedure for preparing the sample using the coated GZH66 enriched microbeads was essentially the same as in example 5, except that only the photo-cutting reaction was performed in S6, specifically, the enriched microbeads were resuspended in 60% aqueous methanol solution, illuminated in a light-transmissive carrier for 30min at 365nm, and the entire procedure was protected from light prior to illumination. The concentration of the methanol aqueous solution can be selected between 50 and 70 percent according to the needs, and the illumination time can be selected within 20 to 50 minutes according to the actual needs.

Applicants have used Acid Cut (AC) and Photo Cut (PC) linker in parallel to enrich for probe-tagged peptides in 200 μg whole proteome and the mass spectrometry results are shown in fig. 6. Using AC linker, the applicant could identify on average 6247 probe-tagged peptide fragments in two replicates, a number slightly less than the previous identification, possibly due to experimental batch effects. With PC linker, applicant could identify 2519 probe-tagged peptide fragments on average in two replicates, the number of which was far lower than that of AC linker, but applicant's preliminary data could prove that the superTOP-ABPP procedure was applicable to linkers of different cutting modes, and subsequently could optimize the experimental procedure according to the linker of different cutting modes, improving the coverage of the sites.

In summary, the applicant developed a solid phase medium carrying an azide group and having an acid or photo-cleavable linker in the middle for efficient enrichment of probe-tagged peptide fragments or proteins, designated superTOP-ABPP. Compared with the traditional TOP-ABPP sample preparation flow, the superTOP-ABPP takes shorter time, has lower experimental cost and can identify more probe-labeled peptide fragments. Because the superTOP-ABPP strategy has higher enrichment efficiency, high coverage identification of probe mark sites can be realized from a trace amount of samples, and the application of chemical proteomics in rare samples (such as rare tissues or primary cultured cells) can be promoted. The efficient enrichment strategy also ensures the quantitative accuracy of superTOP-ABPP, which can reach levels similar to the SILAC and isoTOP-ABPP strategies as quantitative gold standards from the current data. The method has good universality for different bio-orthogonal groups, for example, the method can be used for enriching cyclooctyne probe marked substrates. Also, if the solid phase medium prepared into alkynyl is used for enriching the free azide probe labeled protein in the solution, the method has better effect. According to the literature investigation result of the applicant, the research uses the cleavable medium loaded with the azide group to prepare a TOP-ABPP sample for the first time, and besides the acid cutting linker, the applicant also tries to complete the whole experimental procedure by using the photo cutting linker, so that the development procedure of the applicant can be compatible with the linkers with different cutting modes.

Example 6: the remainder was the same as in example 5 except that:

the alkynyl probe in the S1 is a lysine targeting probe STPyne, the adding amount of the STPyne in the whole cell lysate is 1000 mu M, and the method is applicable to all biological orthogonal probes such as alkynyl, cyclooctyne, azide probes and the like.

The final concentration of dithiothreitol in the S2 is 5mM, and the reaction condition of the dithiothreitol is 30 ℃ for 40min; the content of SDS in the phosphate buffer solution is 1.0%;

the final concentration of the iodoacetamide in the S3 is 10mM, and the light-shielding reaction condition is that the light-shielding reaction is carried out for 40min at 30 ℃;

and in the step S4, a pancreatin enzyme digestion step is omitted.

The protein concentration of the alkynyl probe marked protein liquid in the S5 is 1 mug/mu L;

the concentration of urea in the step S6 is 4M, the acid solution in the acid cutting reaction is 3% acetic acid aqueous solution, and the time is 120min. Acetic acid may be replaced by other organic acids such as propionic acid.

The applicant develops a process which can be also used for identifying the STPyne probe marking target point, and shows that the method is compatible with different types of probes and has better universality.

Example 7: the arginine site for high reactivity of MGO was identified by the SuperTOP-ABPP method, except that the procedure was as in example 5, except that:

the alkynyl probe in the S1 is an arginine reactive probe MGOyne, and the addition amount of the MGOyne in the whole cell lysate is 200 mu M in final concentration.

The final concentration of dithiothreitol in the S2 is 15mM, and the reaction condition of the dithiothreitol is 40 ℃ for 20min; the content of SDS in the phosphate buffer solution is 1.5%;

the final concentration of the iodoacetamide in the S3 is 30mM, and the light-shielding reaction condition is that the light-shielding reaction is carried out for 20min at 40 ℃;

and in the step S4, a pancreatin enzyme digestion step is omitted.

The protein concentration of the alkynyl probe marked protein liquid in the S5 is 5 mug/mu L.

The concentration of urea in the step S6 is 6M, the acid solution in the acid cutting reaction is 1% hydrochloric acid aqueous solution, and the time is 30min.

Methylglyoxal (MGO) is an important product in the tricarboxylic acid cycle, and the MGO molecule can react with nucleophilic amino acids and affect the function of proteins. Because of the unstable structure of MGO, cross-linking readily occurs, and there is currently no systematic understanding of the modified substrates of MGO. The superTOP-ABPP technology developed in the application can effectively shorten the sample preparation time, and can also be used for identifying the MGO modified substrate site. The MCF-7 whole cell proteome was treated with 100. Mu.M MGO or water (control) for 1.5 hours in this example, and then labeled with 200. Mu.M MGO-alkyne probe for 1 hour. The labeled samples were prepared by the superTOP-ABPP workflow and then analyzed by LC-MS/MS (panel a in fig. 13). In this example, two biological replicates were performed with good agreement between the replicates, and the quantitative coefficient of variation of the peptide fragments was within 20% around 90% (panel b in fig. 13). It is speculated that the signal intensity of the sites of high reactivity of the MGO in the MGO-treated group may be significantly reduced. Log2 median for the MGO/control was-0.324 (panel c in fig. 13), also demonstrating that the MGO treatment attenuated the marking of MGOyne, consistent with expectations. Notably, "GR x DQGPNVCALQILGTK" from the period protein was below 0.07 for both replicates (d plot in fig. 13), indicating a likely higher reactivity with MGO.

The foregoing detailed description has been provided for the purposes of illustration in connection with specific embodiments and exemplary examples, but such description is not to be construed as limiting the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications and improvements may be made to the technical solution of the present application and its embodiments without departing from the spirit and scope of the present application, and these all fall within the scope of the present application. The scope of the application is defined by the appended claims.

Claims (10)

1. A rapid and efficient method for preparing a chemical proteomic sample, comprising the steps of:

s1, adding an alkynyl probe into whole cell lysate for marking, adding methanol chloroform to precipitate protein into the marked whole cell lysate, and centrifuging to discard supernatant;

s2, washing the protein precipitate with precooled methanol, re-suspending the protein precipitate with a phosphate buffer solution containing SDS, and adding dithiothreitol for reaction to obtain a mixed solution I;

s3, adding iodoacetamide into the first mixed solution for light-shielding reaction to obtain a second mixed solution;

s4, adding methanol and chloroform into the mixed solution II for precipitation, washing the precipitate with precooled methanol, and then re-suspending the precipitate with PBS to obtain alkynyl probe marked protein liquid;

s5, adding enrichment microbeads into the alkynyl probe marked protein liquid, and fixing protein or peptide fragments marked by the alkynyl probe on the surface of the enrichment microbeads through CuAAC; the enrichment microbeads are microbeads coated with cleavable compounds, the cleavable compounds comprise cleavable groups, one end of each cleavable group is connected with an amino group, the other end of each cleavable group is connected with an azide group, and the cleavable compounds are combined on the surfaces of the enrichment microbeads through amino coating; the azide group and the alkynyl in the alkynyl probe undergo click chemical reaction;

s6, centrifuging after the click chemistry reaction is finished, discarding the supernatant, taking the enrichment microbeads, washing the enrichment microbeads with urea aqueous solution, PBS and water in sequence, cutting off cleavable groups by utilizing a cutting reaction after washing, releasing protein or peptide fragments from the enrichment microbeads in a free manner, centrifuging after the cutting reaction is finished, taking the supernatant, and spin-drying the supernatant in vacuum to obtain the proteomics sample.

2. The method for preparing a rapid and efficient chemical proteomics sample according to claim 1, wherein the cleavage reaction is an acid cleavage reaction or a photo cleavage reaction, and the acid cleavage reaction is a reaction of resuspending enriched microbeads with labeled proteins or peptide fragments bound on the surface in an acid solution; the acid is at least one of formic acid, acetic acid, propionic acid or hydrochloric acid;

the photocleavage reaction is to resuspend the enriched microbeads with the marked proteins or peptide fragments bound on the surface in methanol solution, and then give light to carry out the photocleavage reaction.

3. The method for preparing a rapid and efficient chemical proteomics sample according to claim 2, wherein when the cleavage reaction is an acid cleavage reaction, the cleavable compound is an acid cleavage linker compound, and the structure of the cleavable compound is as shown in formula (I):

4. the method for preparing a rapid and efficient chemical proteomics sample according to claim 2, wherein when the cleavage reaction is a photo-cleavage reaction, the cleavable compound is a photo-cleavage linker compound having a structure as shown in formula (II):

5. the method of claim 3 or 4, wherein the microbeads are NHS-modified agarose beads.

6. The method of claim 5, wherein the enriched microbeads are prepared as follows:

(1) Preparing a 40mM stock solution of an acid cutting linker compound or a light cutting linker compound by using DMSO;

(2) Taking a proper amount of NHS modified agarose beads, ensuring that 50 mu L of solid medium is used for each reaction, washing the NHS modified agarose beads by precooled hydrochloric acid, re-suspending the NHS modified agarose beads in 10mL of Triton PBS solution, adding a stock solution, coating and incubating; the volume ratio of the NHS modified agarose bead heavy suspension to the stock solution is 6-10: 1, a step of;

(3) Centrifuging after incubation, discarding supernatant, and sealing with ethanolamine solution;

(4) Centrifuging, discarding the supernatant, washing with PBS, and re-suspending the solid in glycerol PBS solution to obtain the enriched microbeads.

7. The rapid and efficient chemical proteomic sample preparation method according to claim 6, wherein the concentration of the pre-cooled hydrochloric acid is 1mM, the Triton PBS solution is 0.1% Triton PBS solution, and the concentration of the ethanolamine solution is 0.1M; the ethanol amine solution is blocked at room temperature for 6 hours or at 4 ℃ overnight; the glycerol PBS solution is 30% glycerol PBS solution.

8. A rapid and efficient chemical proteomics sample preparation method according to claim 2, characterized in that,

the alkynyl probe in the S1 is a bio-orthogonal probe, and the final concentration of the alkynyl probe in the whole cell lysate is 1-1000 mu M;

the final concentration of dithiothreitol in the S2 is 5-15 mM, and the reaction condition of the dithiothreitol is 30-40 ℃ for 20-40 min; the content of SDS in the phosphate buffer solution is 1.0% -1.5%;

the final concentration of the iodoacetamide in the S3 is 10-30 mM, and the light-shielding reaction condition is 30-40 ℃ and the light-shielding reaction is carried out for 20-40 min;

the protein concentration of the alkynyl probe marked protein liquid in the S5 is 1-5 mug/mu L, and the CuAAC is prepared by adding 50 mu L of enrichment microbeads, 40 mu L of 25mM Cu-BTTAA stock solution and 120 mu L of 5% sodium ascorbate into each 1mL of alkynyl probe marked protein liquid, and reacting for 2 hours at 25-29 ℃;

the concentration of urea in the S6 is 4-8M.

9. The rapid and efficient chemical proteomics sample preparation method according to claim 8, wherein the alkynyl probe marked protein solution is subjected to pancreatin digestion for 4-12 h to obtain an alkynyl probe marked peptide solution before the step S5, and the alkynyl probe marked peptide solution is subjected to CuAAC reaction with the enrichment microbeads.

10. The method for preparing a rapid and efficient chemical proteomics sample according to claim 8, wherein the acid cleavage reaction is to add 200 μl of 2% formic acid aqueous solution into each 50 μl of enriched microbeads, and the reaction is carried out for 30-120 min after resuspension;

the photo-cutting reaction is to re-suspend the enriched microbeads with 50-70% methanol aqueous solution, and light at 365nm in a light-transmitting carrier for 20-50 min.

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