A fast and efficient method for chemical proteomics sample preparation

Abstract

The application discloses a fast and efficient chemical proteomics sample preparation method, relates to the field of proteomics, and designs and synthesizes agarose microspheres coated with a cleavable linker compound. The free end of the cleavable linker compound contains an azido group, which has good compatibility with existing alkyne probes. Through one-step efficient click chemistry reaction, the probe-labeled peptide segment can be specifically captured on the surface of the agarose microspheres. Since the agarose microspheres and the peptide segment are covalently connected, very strong denaturants or surfactants can be used to wash the medium to remove non-specific adsorption as much as possible. Finally, the probe-labeled peptide segment can be efficiently dissociated from the agarose microspheres through cleavage treatment. The sample preparation method in the application simplifies the cumbersome "click chemistry and streptavidin enrichment" into one-step fast "click chemistry" reaction, and greatly simplifies the operation process.

Description

Technical Field

[0001] This invention relates to the field of proteomics, and more particularly to a rapid and efficient method for preparing chemical proteomics samples. Background Technology

[0002] Activity-based protein profiling (ABPP) 1 ABPP technology provides an efficient analytical platform for the global characterization of protein function. In the ABPP technology system, different chemical probes can specifically capture enzyme active sites, protein functional sites, and post-translational modifications. Combined with affinity enrichment and mass spectrometry analysis, this enables the detection of probe-labeled proteins.

[0003] Chemical probes typically contain a "reactive group" that reacts with a protein's functional site and a "reporter group" that can be used for enrichment or detection. The "reporter group" can also be replaced with a bioorthogonal group (such as an alkynyl group), and then linked to an enrichable tag via a click-through chemical reaction. Introducing a cleavable linker (such as the classic TEV cleavage linker) into the enrichment tag allows the probe-tagged peptide to be specifically dissociated from the affinity medium. This strategy, derived from ABPP, is called Tandem Orthogonal Proteolysis Activity-based Protein Profiling (TOP-ABPP). 2 ).

[0004] Subsequent studies have further expanded the application of TOP-ABPP, mainly in three aspects: 1. The introduction of isotope labeling strategies has enabled the specific quantification of probe-labeled peptides across different groups; 2. The development of various types of cleavable linkers to improve the dissociation efficiency of probe-labeled peptides; 3. The development of different chemical reactive groups to capture different reactive residues, enzyme active sites, and post-translational modifications. To date, TOP-ABPP has achieved widespread application in drug target discovery and lead compound screening.

[0005] However, the traditional TOP-ABPP sample preparation process is lengthy, typically requiring 2-3 days or even longer to prepare a batch of samples. Figure 1The complex operation procedure introduces more variables, which affects the quantitative reproducibility between samples and limits the sample preparation throughput. Meanwhile, the enrichment tag with cleavable linker and commercialized streptavidin enrichment medium are relatively expensive, leading to high experimental cost. To solve the above problems, the applicant hopes to develop a more simplified, time-saving and reagent consumption cost-saving sample preparation procedure of TOP-ABPP.

[0006] The references are as follows:

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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. SUMMARY

[0014] The present application provides a chemical proteomics sample preparation method, which solves the problems of long traditional TOP-ABPP sample preparation process, complicated method and high cost.

[0015] To solve the above problems, the present application provides the following technical solutions:

[0016] A fast and efficient chemical proteomics sample preparation method, comprising the following steps:

[0017] S1, add an alkyne probe to the whole cell lysate for labeling, add methanol chloroform to the labeled whole cell lysate to precipitate proteins, and centrifuge to discard the supernatant;

[0018] S2, after washing the protein precipitate with pre-cooled methanol, resuspend it with a phosphate buffer containing SDS, and then add dithiothreitol to obtain a mixed solution one;

[0019] S3, add iodacetamide to the mixed solution one to obtain a mixed solution two under light protection;

[0020] S4, add methanol chloroform to the mixed solution two for precipitation, wash the precipitate with pre-cooled methanol, and resuspend the precipitate with PBS to obtain an alkyne probe labeled protein solution;

[0021] S5, add enrichment microbeads to the alkyne probe labeled protein solution, and fix the alkyne probe labeled proteins or peptide segments on the surface of the enrichment microbeads through CuAAC; the enrichment microbeads are microbeads coated with a cleavable compound, the cleavable compound comprises a cleavable group, one end of the cleavable group is connected with an amino group, and the other end is connected with an azide group, the cleavable compound is combined on the surface of the enrichment microbeads through the amino group, as shown in (a) of FIG. Figure 1 The azide group reacts with the alkyne group in the alkyne probe through click chemistry;

[0022] S6, after the completion of the click chemistry reaction, centrifuge to discard the supernatant, wash the enrichment microbeads with urea aqueous solution, PBS and water in sequence, cut the cleavable group after washing to release the proteins or peptide segments from the enrichment microbeads, centrifuge to obtain the supernatant after the completion of the cutting reaction, and then vacuum spin dry the supernatant to obtain a proteomics sample.

[0023] Preferably, the cutting reaction is an acid cutting reaction or a light cutting reaction, the acid cutting reaction is to resuspend the enrichment microbeads with the labeled proteins or peptide segments on the surface in an acid solution for reaction, and the acid is at least one of formic acid, acetic acid, propionic acid or hydrochloric acid;

[0024] The light cutting reaction is to resuspend the enrichment microbeads with the labeled proteins or peptide segments on the surface in a methanol solution, and then give light to perform the cutting reaction.

[0025] Preferably, when the cutting reaction is an acid cutting reaction, the cleavable compound is an acid cutting linker compound, and its structure is shown in formula (I):

[0026]

[0027] Preferably, when the cleavage reaction is photocleavage, the cleavable compound is a photocleavable linker compound, which has the structure shown in formula (II):

[0028]

[0029] Preferably, the enriched microbeads are prepared as follows:

[0030] (1) The acid-cleavable linker compound or the photocleavable linker compound is prepared into a 40 mM stock solution with DMSO;

[0031] (2) An appropriate amount of NHS-modified agarose beads is taken to ensure 50 μL of solid medium for each reaction, and the NHS-modified agarose beads are pre-cooled with hydrochloric acid. The NHS-modified agarose beads are resuspended in 10 mL of Triton PBS solution, and the stock solution is added for coating incubation. The volume ratio of the NHS-modified agarose bead resuspension to the stock solution is 6-10:1;

[0032] (3) After incubation, the supernatant is centrifuged and discarded, and then ethanolamine solution is used for blocking;

[0033] (4) The supernatant is centrifuged and discarded, and then the solid is resuspended in glycerol PBS solution after PBS washing to obtain the enriched microbeads.

[0034] Preferably, the concentration of the pre-cooled hydrochloric acid is 1 mM, the Triton PBS solution is 0.1% Triton PBS solution, and the concentration of the ethanolamine solution is 0.1 M. The ethanolamine solution is blocked at room temperature for 6 hours or at 4°C overnight. The glycerol PBS solution is 30% glycerol PBS solution.

[0035] Preferably, the alkynyl probe in S1 is a biorthogonal probe, and the final concentration of the alkynyl probe in the whole cell lysate is 1-1000 μM;

[0036] The final concentration of dithiothreitol in S2 is 5-15 mM, and the reaction conditions of dithiothreitol are 30-40°C for 20-40 min. The content of SDS in the phosphate buffer is 1.0%-1.5%;

[0037] The final concentration of iodine acetamide in S3 is 10-30 mM, and the light-avoiding reaction conditions are 30-40°C for 20-40 min in the dark;

[0038] The protein concentration of the alkynyl probe-labeled protein solution in S5 is 1-5 μg / μL, and the CuAAC is 50 μL of enriched microbeads, 40 μL of 25 mM Cu-BTTAA stock solution, and 120 μL of 5% sodium ascorbate per 1 mL of alkynyl probe-labeled protein solution, and the reaction is carried out at 25-29°C for 2 hours;

[0039] The urea concentration in S6 is 4-8 M.

[0040] Preferably, the alkyne probe-labeled protein solution is subjected to trypsin digestion for 4-12 hours to obtain an alkyne probe-labeled peptide segment solution, and the alkyne probe-labeled peptide segment solution is subjected to CuAAC reaction with the enrichment microbeads.

[0041] Preferably, the acid cleavage reaction is adding 200 μL of 2% formic acid aqueous solution to 50 μL of the enrichment microbeads, and resuspending the enrichment microbeads for 30-120 minutes.

[0042] The light cleavage reaction is resuspending the enrichment microbeads with 50-70% methanol aqueous solution, and irradiating the enrichment microbeads with 365 nm light in a light-transmitting carrier for 20-50 minutes.

[0043] The application has the following beneficial effects:

[0044] In the application, an agarose microsphere coated with a cleavable linker compound is designed and synthesized, and the free end of the cleavable linker compound contains an azide group, which has good compatibility with the alkyne probe used in the existing TOP-ABPP experiment. Through one-step efficient click chemistry reaction, the probe-labeled peptide segment can be specifically captured on the surface of the agarose microsphere, as shown in FIG. c of Figure 1 Since the agarose microsphere and the peptide segment are covalently connected, a very strong denaturant or surfactant can be used to wash the medium to remove as much non-specific adsorption as possible, and finally the probe-labeled peptide segment can be efficiently dissociated from the agarose microsphere through acid or light cleavage treatment. The newly developed TOP-ABPP sample preparation process simplifies the tedious "click chemistry and streptavidin enrichment" to one-step rapid "click chemistry" reaction, greatly simplifying the operation process. Compared with the traditional TOP-ABPP, the operation time of the superTOP-ABPP of the application can be reduced to 9 hours, as shown in FIG. d of Figure 1 which can complete the preparation of the sample within one day. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the technical solutions of the application, the following will briefly introduce the drawings needed in the embodiments. Obviously, for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0046] Figure 1Figure (a) is a flow chart of the preparation of agarose beads coated with cleavable linker compounds; Figure (b) is a flow chart of the preparation of a conventional TOP-ABPP sample; Figure (c) is a flow chart of the preparation of a superTOP-ABPP sample; and Figure (d) is a comparison of the time consumption of the conventional TOP-ABPP and superTOP-ABPP strategies.

[0047] Figure 2 Figure (a) and (b) are bar charts showing the number of probe modification sites identified in the TOP-ABPP sample and the superTOP-ABPP sample, respectively. Figure (c) is a comparison of the signal intensity of the peptides in the superTOP-ABPP sample and the TOP-ABPP sample.

[0048] Figure 3 Figure (a) is a mass spectrometry detection result of a TOP-ABPP micro-sample; Figure (b) is a mass spectrometry detection result of a superTOP-ABPP micro-sample; Figure (c) is a comparison of the analysis results obtained by the two sample preparation methods; and Figure (d) is a comparison of the cost of the two sample preparation methods.

[0049] Figure 4 Figure (a) is a standard deviation of the sample enriched first and then combined; Figure (c) is a standard deviation of the sample combined first and then enriched; Figure (b) is a linear relationship between the detected value and the theoretical value of the light-heavy ratio of the sample enriched first and then combined; and Figure (d) is a linear relationship between the detected value and the theoretical value of the light-heavy ratio of the sample combined first and then enriched.

[0050] Figure 5 Figure (a) is a distribution of the peptide intensity ratio under 100 μΜ and 10 μΜ probe labeling; Figure (b) is the number of high-activity cysteines identified among three replicates; and Figure (c) is a correlation analysis of the quantification; and Figure (d) is that the known high-activity cysteine sites can be effectively identified by superTOP-ABPP, and the results have a good correlation compared with the results obtained by the conventional isoTOP-ABPP.

[0051] Figure 6Efficiency comparison of acid-cleavable and photocleavable linker identification. (a) is the mass spectrometry detection result of the sample prepared by using the microbeads coupled with acid-cleavable linker compound for enrichment; (b) is the mass spectrometry detection result of the sample prepared by using the microbeads coupled with photocleavable linker compound for enrichment; (c) is a statistical diagram of identification efficiency of two different enrichment microbeads.

[0052] Figure 7 CY74 compound 1 H NMR spectrum.

[0053] Figure 8 CY75 compound 1 H NMR spectrum.

[0054] Figure 9 CY62 compound 1 H NMR spectrum.

[0055] Figure 10 CY64 compound 1 H NMR spectrum.

[0056] Figure 11 CY66 compound 1 H NMR spectrum.

[0057] Figure 12 Comparison of identification results of traditional TOP-ABPP and superTOP-ABPP using enrichment microbeads when different probe-labeled proteins; (a) is lysine-reactive STPyne probe and its labeled identification result; (b) is cysteine-reactive iodoacetamide alkynyl probe IAyne and its labeled identification result. In the figure, azide beads represent superTOP-ABPP samples prepared by using enrichment microbeads.

[0058] Figure 13 Data of MGOyne target identification by superTOP-ABPP data. (a) is a flowchart of the whole experimental operation, and MGO is used to compete with MGOyne to screen sites whose MGOyne labeling is obviously weakened under MGO treatment as MGO-sensitive sites; (b) is the coefficient of variation distribution of quantitative peptides between repeats; (c) is the signal intensity ratio distribution of peptides under + / - MGO treatment; (d) is the correlation of peptide signal intensity ratio between two repeats. DETAILED DESCRIPTION

[0059] The specific embodiments of the present application are described herein. It should be clear, however, that the description of the embodiments is just a description of some of the embodiments of the present application, and is not meant to be an exhaustive list of the embodiments of the present application. Based on the embodiments described herein, one of ordinary skill in the art would be able to derive other embodiments of the present application without undue experimentation.

[0060] Example 1: Synthesis of acid cleavable linker compound CY75

[0061]

[0062] Reagents and conditions in the synthesis reaction: (a) NaN3, 0-65 °C, overnight, 90%; (b) Ph2SiCl2, TEA, 0 °C-rt, 36 h, 47.5%; (c) 20% piperidine, 0 °C-rt, 21%.

[0063] Synthesis of compound CY72

[0064] Dissolve 6-bromo-1-hexanol (0.96 mL, 6 mmol) in a solution of super dry DMF (15 mL) and cool to 0 °C in an ice bath. Slowly add sodium azide (0.96 g, 12 mmol, 2.0 equiv.). Slowly heat the mixture to 65 °C and stir overnight. After cooling to room temperature, filter, add saturated brine (30 mL), and extract with ethyl acetate (20 mL x 3). Dry the combined organic phases over anhydrous Na2SO4, filter, and vacuum distill to give the crude product.

[0065] Synthesis of compound CY74

[0066] Add Ph2SiCl2(630 μL, 3.0 mmol) and Et3N (832 μL, 6.0 mmol) to 15 mL of dichloromethane in an ice bath. Slowly add a solution of compound 72 (143 mg, 1.0 mmol) in dichloromethane (1.5 mL) dropwise, then slowly warm the reaction to room temperature. After 12 h, add 73 (2034 mg, 6.0 mmol) and continue stirring for 24 h. Extract with dichloromethane (20 mL x 3), dry the combined organic phases over anhydrous Na2SO4, and concentrate under vacuum in a water bath below 30 °C. Purify by column chromatography (PE:EA = 10:1 to 5:1) to give 74 as a white solid (315 mg, 47.5%). As shown in Figure 7 1 ​H NMR (400 MHz, Chloroform-d) δ 7.80 - 7.53 (m, 8H), 7.46 - 7.26 (m, 10H), 4.73 (s, 1H), 4.40 (d, J = 6.8 Hz, 2H), 4.21 (t, J = 6.7 Hz, 1H), 3.77 (t, J = 6.4 Hz, 3H), 3.23 - 3.15 (m, 4H), 1.68 - 1.24 (m, 17H). 13 C NMR (101 MHz, Chloroform-d) δ 156.41, 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 N4O4Si[M+H] + 663.33611, found 663.33652.

[0067] Synthesis of compound CY75

[0068] Compound 74 (100 mg, 0.15 mmol) was added to 10 mL of dichloromethane under ice bath, and 20% piperidine (152 μL) was added dropwise. After stirring for 30 min, the reaction was raised to room temperature. After TLC detection of the end of the reaction, dichloromethane was extracted (20 mL x 3), the organic layers were combined, washed twice with saturated brine, and dried over anhydrous Na2SO4. Filtration, solvent concentration under vacuum in a 30 °C water bath, and purification by preparative thin layer chromatography (CH2Cl2:MeOH = 20:1) gave 75 (14 mg, 21%) as a light yellow solid. As shown in Figure 8 , 1 H NMR (400 MHz, Methanol-d4) δ 7.67 - 7.57 (m, 4H), 7.47 - 7.31 (m, 6H), 3.79 (q, J = 6.3 Hz, 2H), 3.62 - 3.53 (m, 1H), 3.25 (t, J = 6.8 Hz, 1H), 2.93 - 2.85 (m, 1H), 1.69 - 1.51 (m, 6H), 1.47 - 1.22 (m, 12H), 0.90 (t, J = 6.8 Hz, 1H). 13C NMR (101 MHz, Methanol-d4) δ 134.50, 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 N4O2Si[M+H] + 441.26803, found 441.26795.

[0069] Example 2: Synthesis of photocleavable linker compound GZH66

[0070]

[0071] Reagents and conditions: (a) NaN3, 0-90 °C, overnight, 55.9%; (b) i) DCC, Et3N, NHS; ii) Boc-ethylenediamine, 33.9%; (c) 62, DSC, Et3N; (d) TEA, 0 °C, 6 h, 25.9%.

[0072] Synthesis of compound 62

[0073] Dissolve 3-bromopropylamine hydrobromide (8.4 g, 38.7 mmol) in water, ice bath, cool to 0 °C. Slowly add sodium azide (8.5 g, 130.8 mmol, 3.4 eq). Slowly heat the mixture to 90 °C, stir overnight. After cooling to room temperature, filter, add saturated brine (30 mL), extract with ethyl acetate (50 mL x 3). Dry the combined organic phase over anhydrous Na2SO4, filter and distill under vacuum to get the crude product. Purify the product by flash chromatography using 10% methanol in dichloromethane to get 62 (1.0 g, 3.3 mmol, 8.4% yield). Figure 9 As shown, 1 H NMR (400 MHz, CDCl3) δ 3.38 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.8 Hz, 2H), 1.73 (p, J = 6.8 Hz, 2H), 1.18 (s, 2H).

[0074] Synthesis of compound 64

[0075] i) Add 63 (1.0 g, 3.3 mmol) to 25 mL dichloromethane under ice bath, add NHS (760.0 mg, 6.6 mmol) and Et3N (1.0 g, 10.0 mmol). Add DCC (825.2 mg, 4.0 mmol), slowly recover to room temperature, stir overnight. Filter.

[0076] ii) To the filtrate from i), Boc-ethylenediamine (1.1 g, 6.6 mmol) was added and stirred at room temperature overnight. It was washed with saturated brine three times and the product was purified by column chromatography with CH2Cl2:MeOH = 40:1 as eluent. 500 mg of light yellow product was obtained after rotary evaporation. The product was used directly in the next step. Figure 10 as shown, 1 H NMR (400 MHz, MeOD) δ 7.61 (s, 1H), 7.16 (s, 1H), 6.26 (q, J = 5.7 Hz, 1H), 4.10 (q, J = 3.4 Hz, 2H), 3.95 (s, 3H), 3.83 (t, J = 5.7 Hz, 1H), 3.55 (t, J = 5.7 Hz, 1H), 3.47 (t, J = 5.8 Hz, 1H), 3.13 (t, J = 6.5 Hz, 2H), 3.07 (t, J = 5.5 Hz, 1H), 2.63 (s, 1H), 2.50 - 2.40 (m, 4H), 2.15 - 2.07 (m, 2H), 1.69 (dd, J = 13.2, 6.6 Hz, 2H), 1.58 (d, J = 6.3 Hz, 3H).

[0077] Synthesis of compound 65

[0078] DSC (89.6 mg, 0.35 mmol) and Et3N (70.8 mg, 0.70 mmol) were added to 10 mL DMF under ice bath condition, 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. Rotary evaporation and purification by preparative thin layer chromatography (CH2Cl2:MeOH = 20:1) gave crude 65 which was used directly in the next step.

[0079] Synthesis of compound 66

[0080] The crude 65 was dissolved in 10 mL CH2Cl2under ice bath condition. 1 mL trifluoroacetic acid was added and stirred for 6 hours. Purification by preparative thin layer chromatography (CH2Cl2:MeOH = 5:1). As shown, Figure 11 as shown, 1 H NMR (400 MHz, MeOD) δ 7.61 (s, 1H), 7.16 (s, 1H), 6.26 (q, J = 5.7 Hz, 1H), 4.10 (q, J = 3.4 Hz, 2H), 3.95 (s, 3H), 3.83 (t, J = 5.7 Hz, 1H), 3.55 (t, J = 5.7 Hz, 1H), 3.47 (t, J = 5.8 Hz, 1H), 3.13 (t, J = 6.5 Hz, 2H), 3.07 (t, J = 5.5 Hz, 1H), 2.63 (s, 1H), 2.50 - 2.40 (m, 4H), 2.15 - 2.07 (m, 2H), 1.69 (dd, J = 13.2, 6.6 Hz, 2H), 1.58 (d, J = 6.3 Hz, 3H).

[0081] Example 3: Preparation of enrichment beads with agarose beads coupled cleavable linker compounds, the specific steps are as follows:

[0082] 1. CY75 synthesized in Example 1 or GZH66 synthesized in Example 2 is prepared into a 40 mM stock solution with DMSO. GZH66 is operated in the dark during the entire coating process.

[0083] 2. Take an appropriate amount of NHS modified agarose beads, hereinafter referred to as NHS beads (cytiva, item number 17037137), to ensure 50 μL of solid medium for each reaction. After washing the NHS beads with 1 mM ice-cold hydrochloric acid 3 times, resuspend the NHS beads in 10 mL of 0.1% Triton PBS solution. Add 250 μL of CY75 or GZH66 stock solution for every 2 mL of NHS modified agarose bead suspension, and incubate at room temperature for 6 hours or at 4°C overnight. The volume ratio of the NHS modified agarose bead suspension to the stock solution can be selected between 6:1 and 10:1 according to the reaction needs.

[0084] 3. Centrifuge and discard the supernatant, and block the excess NHS group with 0.1 M ethanolamine solution at room temperature for 6 hours or at 4°C overnight to obtain agarose beads coated with CY75 or GZH66.

[0085] 4. Centrifuge and discard the supernatant to obtain the enrichment beads. After washing with PBS for 5 times, resuspend the enrichment beads in 30% glycerol PBS solution, aliquot, and store in a -20°C freezer for standby.

[0086] Example 4: Preparation of samples using TOP-ABPP method, specifically:

[0087] 1. After the whole cell lysate is labeled with a probe, an acid-cleavable biotin tag is connected through CuAAC, and the reaction time is 1 hour.

[0088] 2. The precipitated protein is washed with cold methanol 3 times and resuspended in 1.2% SDS PBS solution.

[0089] 3. Dilute the concentration of SDS to below 0.2%, and then use 100 μL of streptavidin agarose beads, hereinafter referred to as beads (Thermo Scientific, item number 20353) for enrichment, and react at 29 degrees for 4-6 hours.

[0090] 4. After incubation, wash the beads with 5 mL of PBS 3 times, and then transfer to a screw cap tube after washing with 5 mL of water 3 times.

[0091] 5. Centrifuge to discard the supernatant, resuspend the beads in 500 μL 6M urea, add 10 mM dithiothreitol (DTT) and incubate at 37°C for 30 min.

[0092] 6. Add 20 mM iodoacetamide (IAA) and incubate at 35°C for 30 min in the dark.

[0093] 7. Centrifuge to discard the supernatant, resuspend the beads in 200 μL PBS, add trypsin and incubate overnight for enzymatic cleavage.

[0094] Centrifuge to discard the supernatant, wash the beads twice with 1 mL PBS, wash the beads five times with 1 mL water, and then use a formic acid solution to dissociate the probe-labeled peptide fragments from the beads.

[0095] Example 5: Preparation of a cysteine-targeting probe-labeled peptide fragment sample using the super TOP-ABPP method, the super TOP-ABPP sample preparation process is as follows:

[0096] S1. Add iodoacetamide alkyne probes IA-alkyne to the whole cell lysate for labeling, add methanol chloroform to the labeled whole cell lysate to precipitate the protein, centrifuge to discard the supernatant and remove excess probes;

[0097] S2. Wash the protein precipitate twice with pre-cooled methanol, resuspend with a phosphate buffer containing 1.2% SDS, and then add 10 mM dithiothreitol (DTT) and incubate at 37°C for 30 min to obtain a mixed solution one;

[0098] S3. Add iodoacetamide (IAA) to the mixed solution one to a final concentration of 20 mM, incubate at 35°C for 30 min in the dark to obtain a mixed solution two;

[0099] S4. Add methanol chloroform to the mixed solution two for precipitation, wash the precipitate three times with pre-cooled methanol, resuspend the precipitate with PBS, and trypsinize for 4-12 hours to obtain an alkyne probe-labeled peptide fragment solution;

[0100] S5. Add enrichment microbeads to the alkyne probe-labeled peptide fragment solution with a concentration of 2 μg / μL for copper-catalyzed azide and alkyne cycloaddition (CuAAC) reaction, wherein the CuAAC is 50 μL enrichment microbeads, 40 μL 25 mM Cu-BTTAA stock solution, and 120 μL 5% sodium ascorbate per 1 mL alkyne probe-labeled protein solution, and the reaction is carried out at 25-29°C for 2 hours;

[0101] S6. After the reaction is complete, centrifuge and collect the enriched microbeads. Wash the enriched microbeads once with 8M urea, twice with 1mL PBS, and five times with 1mL water. After washing, add 200μL of 2% formic acid aqueous solution to every 50μL of enriched microbeads, resuspend, and react for 60min to dissociate the probe-labeled peptides from the enriched microbeads. After the reaction is complete, centrifuge and collect the supernatant to obtain the proteomics sample.

[0102] This method can be used to enrich probe-labeled peptides after trypsin digestion, and it can also be used to enrich probe-labeled proteins. When enriching probe-labeled proteins, trypsin digestion is not required.

[0103] The applicant conducted a parallel comparison of the identification efficacy of TOP-ABPP and superTOP-ABPP for probe modification sites. The applicant used an iodoacetamide-alkynyl probe (IA-alkyne) that specifically reacts with cysteine ​​to label the proteome, and then processed the samples using both superTOP-ABPP and conventional ABPP procedures. Mass spectrometry results showed that, under the same starting conditions, TOP-ABPP identified 6953 modification sites, while superTOP-ABPP identified 12814 probe modification sites. Figure 2 (a, b). Meanwhile, the applicant found that the median Log2 signal intensity of peptides identified by TOP-ABPP was approximately 21.7, while the median Log2 signal intensity of peptides in the superTOP-ABPP group was 24.1. In other words, the enrichment efficiency of superTOP-ABPP for probe-modified peptides is approximately 5 times that of TOP-ABPP.

[0104] Given the high enrichment efficiency of superTOP-ABPP, the applicant hypothesizes that this strategy can be used for chemical proteomics analysis with fewer samples. In traditional TOP-ABPP experiments, the initial sample amount is 2 mg per sample. Here, the applicant uses 1 / 10 of the traditional TOP-ABPP initial sample amount (200 μg) to test the enrichment sensitivity of superTOP-ABPP. 200 μg of the whole proteome labeled with the IA-alkyne probe was processed using both TOP-ABPP and superTOP-ABPP procedures. Based on the results of mass spectrometry identification (see...),... Figure 3 The TOP-ABPP method identified 4604 probe-labeled peptides, while the superTOP-ABPP method identified 7913 probe-labeled peptides. Figure 3As shown in Figures (a) to (c), the superior peptide identification performance demonstrates the significant application potential of the superTOP-ABPP strategy in the chemical proteomics analysis of trace samples. Furthermore, the applicant compared the experimental costs of superTOP-ABPP and traditional TOP-ABPP: preparing a TOP-ABPP sample costs approximately 61 yuan, while a superTOP-ABPP sample costs only 8 yuan, which is only 13% of the experimental cost of traditional TOP-ABPP. Figure 3 As shown in Figure (d) and Table 1. In summary, compared with traditional sample preparation methods, superTOP-ABPP is a faster, more efficient, and more economical chemical proteomics sample preparation process.

[0105] Table 1. Detailed comparison of TOP-ABPP and superTOP-ABPP in terms of experimental time, reagent cost, and number of identified peptides.

[0106] Comparison with TOP-ABPP

[0107]

[0108] Next, the applicant evaluated the accuracy and dynamic range of the superTOP-ABPP strategy for quantifying probe-labeled peptides. In current quantitative proteomics research, stable isotope labeling by amino acids in cell culture (SILAC) is the gold standard for quantification. Here, SILAC labeling was used to obtain lightly labeled (lysine +0, arginine +0) and heavily labeled (lysine +8, arginine +10) MCF-7 cells. After cell lysis, the cells were labeled with IA-alkyne probes and then enriched using the applicant's proposed strategy, with light-labeled to heavily labeled ratios of 5:1, 2:1, 1:1, 1:2, and 1:5, respectively. After enrichment, samples from different light- and heavily labeled groups were mixed as experimental groups to test the interference of enrichment on quantification. Simultaneously, a control group was set up with pre-mixed light- and heavily labeled samples enriched simultaneously, representing the true proportion of light- and heavily labeled peptides in the samples. Mass spectrometry analysis results showed that the actual light-to-heavy ratio detected in the control group had a good linear relationship with the expected ratio (R0). 2 =0.99)( Figure 4 (Figure b). The light-to-weight ratio detected in the experimental group also showed a good linear relationship with the expected value (R0). 2 =1.00)( Figure 4 (Figure d). Meanwhile, the standard deviations of the samples in the experimental and control groups were similar ( Figure 4Figure 2a, c). These results indicate that the superTOP-ABPP method has similar quantitative accuracy and linear dynamic range as SILAC.

[0109] For quantitative analysis of chemical proteomics, another widely used technique is the isoTOP-ABPP 7 (isotopic Tandem Orthogonal Proteolysis Activity Based Protein Profiling) technique. The isoTOP-ABPP technique was used to quantitatively compare the cysteine signals between high and low concentrations of probe labeling in proteomes, and systematically analyzed the highly active cysteines in cells, providing a very powerful tool for the study of cysteine function. The principle is that highly reactive cysteines will quickly achieve saturation labeling at low probe concentrations. Therefore, if the applicant uses high and low concentrations of probes to label the cysteines in the cell, the signal intensity ratio of highly active cysteines between the two groups should be close to 1 (R:[low]≈1). In this study, the applicant used the superTOP-ABPP combined with reduced dimethylation quantitative method to quantitatively analyze the cysteine group labeled by high and low concentrations of probes, and compared it with the classical isoTOP-ABPP strategy to further test the quantitative accuracy and dynamic range of superTOP-ABPP in highly complex samples. According to the results of mass spectrometry: isoTOP-ABPP quantified 1507 probe-modified peptides in three replicates, and the superTOP-ABPP group quantified 1841 probe-modified peptides Figure 5 Figure 2a, c). These results indicate that the superTOP-ABPP method has similar quantitative accuracy and linear dynamic range as SILAC. Figure 5 Figure 2b). Finally, the superTOP-ABPP strategy quantified a total of 68 highly reactive cysteine sites in three replicates, and the quantitative ratios of these 68 sites showed high consistency between the three replicates (R 2 = 0.87-0.93) (Figure 2c). Figure 5Figure 6C). The above results demonstrated the high reproducibility of superTOP-ABPP strategy in quantification. Among the 68 sites, there are a series of highly reactive cysteine sites well known to the skilled person, such as CKB (C283), PRMT1 (C101), ACAT1 (C126) and GSTO1 (C32). For these known highly active cysteine sites, the isoTOP-ABPP and superTOP-ABPP quantification ratios are quite close (Figure 6D), which again confirmed the quantitative accuracy of superTOP-ABPP strategy in complex samples. Figure 5 Figure 6C). The above results demonstrated the high reproducibility of superTOP-ABPP strategy in quantification. Among the 68 sites, there are a series of highly reactive cysteine sites well known to the skilled person, such as CKB (C283), PRMT1 (C101), ACAT1 (C126) and GSTO1 (C32). For these known highly active cysteine sites, the isoTOP-ABPP and superTOP-ABPP quantification ratios are quite close (Figure 6D), which again confirmed the quantitative accuracy of superTOP-ABPP strategy in complex samples.

[0110] In addition to the acid-cleavable (AC) linker compound, the applicants also synthesized a photo-cleavable (PC) GZH66, which can be coupled to NHS beads through the same procedure for the enrichment of alkyne probe-labeled peptides and proteins. The specific steps for preparing samples using GZH66-coated enrichment microbeads are basically the same as those in Example 5, except that only a photo-cleavage reaction is performed in S6. The photo-cleavage reaction is specifically resuspending the enrichment microbeads in a 60% methanol aqueous solution and irradiating them with 365 nm light for 30 min in a light-transmitting carrier. The operation process before light irradiation is carried out in the dark throughout. The concentration of the methanol aqueous solution can be selected between 50% and 70% as needed, and the light irradiation time can be selected within 20-50 min as needed.

[0111] The applicants used acid-cleavable (AC) and photo-cleavable (PC) linkers to enrich probe-labeled peptides in 200 micrograms of whole proteome in parallel, and the mass spectrometry identification results are shown in Table 1. Figure 6 Using the AC linker, the applicants can identify an average of 6247 probe-labeled peptides in two repeats, which is slightly less than the previous identification results, which may be caused by batch effects of the experiment. Using the PC linker, the applicants can identify an average of 2519 probe-labeled peptides in two repeats, which is much lower than the AC linker, but the applicants' preliminary data can prove that the superTOP-ABPP procedure is applicable to linkers of different cleavage modes, and subsequent experiments can be optimized according to the linkers of different cleavage modes to improve the coverage of sites.

[0112] In summary, the applicant developed a kind of solid phase medium loaded with azido groups and with acid or light cut linkers in the middle, which is used for efficient enrichment of probe-labeled peptides or proteins, and is named superTOP-ABPP. Compared with the traditional TOP-ABPP sample preparation process, superTOP-ABPP takes less time, costs less, and can identify more probe-labeled peptides. Because of the high enrichment efficiency of superTOP-ABPP strategy, high coverage identification of probe-labeled sites can be achieved from very small amounts of samples, which can promote the application of chemical proteomics in rare samples (such as rare tissues or primary cultured cells). The efficient enrichment strategy also ensures the quantitative accuracy of superTOP-ABPP. From the current data, superTOP-ABPP can achieve a level similar to SILAC and isoTOP-ABPP strategies, which are the gold standard for quantification. In this experiment, azido solid phase medium is used to enrich free alkyne probe-labeled peptides or proteins in solution. This method has good universality for different biological orthogonal groups, such as the enrichment of cyclooctyne probe-labeled substrates. Similarly, if the solid phase medium is prepared with alkyne groups to enrich free azido probe-labeled proteins in solution, it should also have good results. According to the applicant's literature research results, this study is the first time to use a cleavable medium loaded with azido groups to prepare TOP-ABPP samples. In addition to acid cut linkers, the applicant also tried to use a light cut linker to complete the entire experimental process, proving that the process developed by the applicant can be compatible with linkers of different cutting methods.

[0113] Example 6: The rest is the same as example 5, except that:

[0114] The alkyne probe in S1 is lysine-targeting probe STPyne, and the addition amount of STPyne in whole cell lysate is 1000 μM of final concentration. This method is suitable for all biological orthogonal probes, such as alkyne, cyclooctyne, azido probe, etc.

[0115] The final concentration of dithiothreitol in S2 is 5 mM, and the reaction condition of dithiothreitol is 30°C for 40 min; the content of SDS in the phosphate buffer is 1.0%;

[0116] The final concentration of iodoacetamide in S3 is 10 mM, and the light-avoiding reaction condition is 30°C for 40 min;

[0117] The trypsin digestion step in S4 is omitted.

[0118] The protein concentration of the alkyne probe-labeled protein solution in S5 is 1 μg / μL;

[0119] The urea concentration in S6 is 4M, the acid solution in the acid cleavage reaction is 3% acetic acid aqueous solution, and the time is 120 min. Acetic acid can also be replaced by propionic acid and other organic acids.

[0120] The applicant developed a process that can also be used for the identification of STPyne probe-labeled targets, indicating that the method is compatible with different types of probes and has good universality.

[0121] Example 7: Identification of MGO-hypersensitive arginine sites by superTOP-ABPP method, the rest are the same as Example 5, the difference is that:

[0122] The alkynyl probe in S1 is the arginine-reactive probe MGOyne, and the addition amount of MGOyne in whole cell lysate is 200 μM of final concentration.

[0123] The final concentration of dithiothreitol in S2 is 15 mM, and the reaction condition of dithiothreitol is 40°C for 20 min; the content of SDS in the phosphate buffer is 1.5%;

[0124] The final concentration of iodoacetamide in S3 is 30 mM, and the light-avoiding reaction condition is 40°C for 20 min in the dark;

[0125] The trypsin enzyme digestion step is omitted in S4.

[0126] The protein concentration of the alkynyl probe-labeled protein solution in S5 is 5 μg / μL.

[0127] The urea concentration in S6 is 6M, the acid solution in the acid cleavage reaction is 1% hydrochloric acid aqueous solution, and the time is 30 min.

[0128] Methylglyoxal (MGO) is an important product in the tricarboxylic acid cycle, and MGO molecules can react with nucleophilic amino acids and affect the function of proteins. Due to the unstable structure of MGO, cross-linking easily occurs, and at present there is a lack of systematic understanding of the modified substrates of MGO. The superTOP-ABPP technology developed in this application can effectively shorten the sample preparation time, and can also be used for the identification of MGO-modified substrate sites. In this example, 100 μM MGO or water (control) was used to treat MCF-7 whole cell proteome for 1.5 hours, and then 200 μM MGO-alkyne probe was used for labeling for 1 hour. The labeled sample was prepared by the superTOP-ABPP workflow, and then analyzed by LC-MS / MS (Figure a). Figure 13 The two biological repeats in this example have good consistency, and the quantitative coefficient of variation of about 90% of the peptides is within 20% ( Figure 13Figure 6C, which also demonstrates that MGO treatment attenuates the MGO- yne signature, as expected. It is notable that the "GR*DQGPNVCALQILGTK" from Periostin protein has a MGO / control ratio below 0.07 in both replicates Figure 13 Figure 6C, which also demonstrates that MGO treatment attenuates the MGO- yne signature, as expected. It is notable that the "GR*DQGPNVCALQILGTK" from Periostin protein has a MGO / control ratio below 0.07 in both replicates Figure 13 Figure 6D, which indicates that it can have a higher reactivity with MGO.

[0129] The above detailed description and exemplary examples of the application are not to be understood as limitations on the present application. Alternative examples of the application not described above are possible and will be apparent to those skilled in the art in view of the above description and the examples. Such alternatives are considered to be within the scope of the present application. The scope of the application is defined by the appended claims, rather than the detailed description and examples given above.

Claims

1. A rapid and efficient method for preparing chemical proteomics samples, characterized in that, It includes the following steps: S1. Add an alkyne probe to the whole cell lysate for labeling. Add methanol and chloroform to the labeled whole cell lysate to precipitate the protein. Centrifuge and discard the supernatant. S2. After washing the protein precipitate with pre-cooled methanol, resuspend it in phosphate buffer containing SDS, and then add dithiothreitol to react and obtain mixture one. S3. Add iodoacetamide to mixture one and react in the dark to obtain mixture two; S4. Methanol and chloroform were added to mixture two for precipitation. The precipitate was washed with pre-cooled methanol and then resuspended in PBS to obtain alkyne probe labeled protein solution. S5. Add enrichment beads to the alkyne probe-labeled protein solution, and use CuAAC to immobilize the alkyne probe-labeled protein or peptide on the surface of the enrichment beads; the enrichment beads are beads coated with a cleavable compound, the cleavable compound containing a cleavable group, one end of the cleavable group being connected to an amino group and the other end being connected to an azide group, the cleavable compound being bound to the surface of the enrichment beads through the amino group coating; the azide group undergoes a click chemical reaction with the alkyne group in the alkyne probe. S6. After the chemical reaction is completed, centrifuge and discard the supernatant. Take the enriched microbeads and wash them sequentially with urea aqueous solution, PBS and water. After washing, use a cleavage reaction to cut the cleavable groups and release the protein or peptide from the enriched microbeads. After the cleavage reaction is completed, centrifuge and take the supernatant. Then, vacuum dry the supernatant to obtain the proteomics sample. The cleavage reaction is an acid cleavage reaction or a photocleavage reaction.

2. The rapid and efficient chemical proteomics sample preparation method according to claim 1, characterized in that, The acid cleavage reaction involves resuspending enriched microbeads with labeled proteins or peptides bound to their surface in an acid solution and reacting them; the acid is at least one of formic acid, acetic acid, propionic acid, or hydrochloric acid. The photocutting reaction involves resuspending enriched microbeads with labeled proteins or peptides on their surface in a methanol solution and then subjecting them to light to induce a cutting reaction.

3. The rapid and efficient chemical proteomics sample preparation method according to claim 2, characterized in that, When the cleavage reaction is an acid cleavage reaction, the cleavable compound is an acid-cleaving linker compound with the structure shown in formula (I):

4. The rapid and efficient chemical proteomics sample preparation method according to claim 2, characterized in that, When the cleavage reaction is a photo-cleavage reaction, the cleavable compound is a photo-cleaving linker compound, the structure of which is shown in formula (II):

5. A rapid and efficient method for preparing chemical proteomics samples according to claim 3 or 4, characterized in that, The microbeads are NHS-modified agarose beads.

6. The rapid and efficient chemical proteomics sample preparation method according to claim 5, characterized in that, The enriched microspheres were prepared by the following method: (1) Prepare a 40 mM stock solution of acid-cut linker compound or optically cut linker compound using DMSO; (2) Take an appropriate amount of NHS-modified agarose beads, ensuring that there is 50 μL of solid medium for each reaction. Wash the NHS-modified agarose beads with pre-cooled hydrochloric acid, then resuspend the NHS-modified agarose beads in 10 mL of Triton PBS solution, add the stock solution, and incubate with coating solution; the volume ratio of the NHS-modified agarose bead resuspension to the stock solution is 6 to 10:

1. (3) After incubation, centrifuge and discard the supernatant, then block with ethanolamine solution; (4) Centrifuge and discard the supernatant. Wash with PBS and resuspend the solid in glycerol PBS solution to obtain enriched microbeads.

7. The rapid and efficient chemical proteomics sample preparation method according to claim 6, characterized in that, The concentration of the pre-cooled hydrochloric acid is 1 mM, the Triton PBS solution is 0.1% Triton PBS solution, and the concentration of the ethanolamine solution is 0.1 M; the ethanolamine solution is sealed at room temperature for 6 hours or at 4°C overnight; the glycerol PBS solution is 30% glycerol PBS solution.

8. The rapid and efficient chemical proteomics sample preparation method according to claim 2, characterized in that, The alkyne probe in S1 is a biologically orthogonal probe, and the final concentration of the alkyne probe in the whole cell lysis buffer is 1–1000 μM. The final concentration of dithiothreitol in S2 is 5-15 mM, and the reaction conditions for dithiothreitol are 30-40℃ for 20-40 min; the content of SDS in the phosphate buffer is 1.0%-1.5%. The final concentration of iodoacetamide in S3 is 10-30 mM, and the reaction conditions are 30-40°C and 20-40 min in the dark. The protein concentration of the alkyne probe-labeled protein solution in S5 is 1-5 μg / μL. The CuAAC is prepared by adding 50 μL of enrichment microbeads, 40 μL of 25 mM Cu-BTTAA stock solution and 120 μL of 5% sodium ascorbate to each 1 mL of alkyne probe-labeled protein solution, and reacting at 25-29℃ for 2 hours. The urea concentration in S6 is 4–8 M.

9. The rapid and efficient chemical proteomics sample preparation method according to claim 8, characterized in that, Before step S5, the alkyne probe-labeled protein solution is digested with trypsin for 4–12 h to obtain an alkyne probe-labeled peptide solution, which is then reacted with enriched microbeads using CuAAC.

10. The rapid and efficient chemical proteomics sample preparation method according to claim 8, characterized in that, The acid shearing reaction is performed by adding 200 μL of 2% formic acid aqueous solution to every 50 μL of enriched microbeads, resuspending and reacting for 30–120 min. The photo-cutting reaction involves resuspending the enriched microspheres in a 50-70% methanol aqueous solution and irradiating them with 365nm light in a transparent carrier for 20-50 minutes.

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