Protein variant complex analysis method based on short-range labeling-controllable release

Abstract

The invention relates to a protein variant compound analysis technology based on a short-range labeling-controllable release technology and a'top-down 'mass spectrum strategy, and designs a short-range labeling probe containing a controllable breaking group and an enriching group to specifically label a target protein variant compound; then enriching on the material by utilizing an enriching group in the probe; releasing a compound enriched on the material by utilizing a controllable fracture group in the probe; furthermore, the target protein variants and the interacting protein variants thereof are accurately identified through a'top-to-bottom 'strategy. According to the invention, the information of the target protein variant and the interacting protein variant thereof can be obtained, and the precise identification of the compound is realized from the protein variant level.

Description

Technical Field

[0001] The present invention relates to a protein variant complex analysis technology based on short-range labeling-controlled release technology and a "top-down" mass spectrometry strategy, which can realize in situ labeling of protein variants in the target protein complex and provide important technical support for the accurate analysis of the target protein complex. Background Art

[0002] Multiple protein molecules produced by a single gene with different sequences and / or chemical modifications are called protein variants. The study of the diversity and abundance of protein variants plays an important role in understanding the functions of biological systems (Smith, LM et al. (2013) Proteoform: a single term describing protein complexity. Nat. Methods 10, 186–187; Van Damme, P. et al. (2014) N-terminal proteomics and ribosome profiling provide a comprehensive view of the alternative translation initiation landscape in mice and men. Mol. Cell. Proteomics 13, 1245–1261. Gawron, D. et al. (2014) The proteome under translational control. Proteomics 14, 2647–2662. "Top-Down" mass spectrometry technology (Top-Down MS) is an ideal method for comprehensive characterization of protein variant sequences and post-translational modifications (Smith LM, Kelleher NL. (2018) Proteoforms as the next proteomics currency. Science. 359, 1106-1107. Bludau I, Aebersold R. (2020) Proteomic and interactomic insights into the molecular basis of cell functional diversity. Nat Rev Mol Cell Biol. 21, 327-340.).In cells, protein variants function by interacting with each other to form protein variant complexes. At present, the analysis of protein variant complexes has been realized based on "top-down" mass spectrometry technology (Albanese P, Tamara S, Saracco G, Scheltema RA, Pagliano C. (2020) How paired PSII-LHCII supercomplexes mediate the stacking of plant thylakoid membranes unveiled by structural mass-spectrometry. Nat Commun. 11, 1361.). However, the methods reported so far mainly purify the protein variant complex in vitro first, and then apply the "top-down" strategy to identify the protein variant complex. Existing methods lack the capture of protein variant complexes in the in situ environment of the cell. At the same time, protein variants with weak or transient interactions are easily lost during the separation of protein variant complexes, making it difficult to obtain true protein variant complexes.

[0003] Proximity labeling is a technique that uses spatially restricted peroxidase to biotinylate neighboring proteins. It has been widely used in "bottom-up" proteomic analysis to analyze protein complexes and identify protein components in specific cell compartments (Ke M, Yuan X, He A, Yu P, Chen W, Shi Y, Hunter T, Zou P, Tian R. (2021) Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics. Nat Commun. 12, 71. Lobingier BT, R, Eichel K, et al. (2017) An Approach to Spatiotemporally Resolve ProteinInteraction Networks in Living Cells. Cell. 169, 350-360.). Currently, there are two main types of labeled probes based on this technology. One is a modified probe based on biotin-phenol (BP) and the phenolic hydroxyl structure on BP, and the other is Alkyne-Phenol (AP) with improved permeability (Li Y, Tian C, Liu K, Zhou Y, Yang J, Zou P. (2020) A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast. Cell Chem Biol. 27, 858-865. Ke M, Yuan X, He A, Yu P, Chen W, Shi Y, Hunter T, Zou P, Tian R. (2021) Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics. Nat Commun. 12, 71.). Based on the protein variant complexes labeled by these two probes, on the one hand, the recovery rate of protein variants after enrichment is low, and on the other hand, the probe molecules introduced on the protein variants increase the complexity of the MS1 and MS / MS spectra of the protein variants, making it difficult to accurately identify the labeled protein variants. Therefore, this technology has not yet been applied to the analysis of protein variant complexes.

[0004] The present invention is a protein variant complex analysis technology based on short-range labeling-controlled release technology and "top-down" mass spectrometry strategy, and establishes a new method for in situ labeling, specific capture and efficient controlled release of protein variant complexes, providing important technical support for the accurate identification of target protein variant complexes. Summary of the invention

[0005] The present invention relates to a protein variant complex analysis technology based on short-range labeling-controlled release technology and "top-down" mass spectrometry strategy, which can realize in situ labeling of target proteins and their interacting protein variants at the living cell level or in the subcellular organelle space, and then use the enrichment groups on the probe to achieve specific enrichment of the target protein and its interacting protein variants, and further controllably release the specifically enriched protein variants through the controllable cleavage groups on the probe, and then use top-down mass spectrometry technology to accurately identify the target protein and its interacting protein variants, thereby achieving accurate analysis of the target protein variant complex composition.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] (1) Proximity labeling probes containing controllable cleavage groups and enrichment groups: one end of the probe is biotin, the other end is a phenol group that can be catalyzed by peroxidase, and the alkyl chain as the connecting arm contains a cleavable group in the sulfonyl-amide bond;

[0008] (2) Cell culture: Select bacteria, fungi or cell lines expressing the target protein in subcellular organelles or whole cells and fused with horseradish peroxidase, and culture to 80-90% density.

[0009] (3) Preparation of probe solution: Use one or more organic solutions selected from organic alcohols, organic acids, dimethyl sulfoxide, and dimethylformamide to prepare a probe solution with a concentration of 100 mM to 1 M; (4) Preparation of labeling culture medium solution: Dilute the breakable specific enrichment probe mother solution to a range of 1 μM to 1 M using one of the cell culture media such as DMEM or MEM or R1640, and incubate in bacteria, fungi or cells at a temperature of 37°C and a carbon dioxide concentration of 5% for 30 min to 2 h.

[0010] (5) Horseradish peroxidase-catalyzed proximity labeling: Add a 0.5-10 mM hydrogen peroxide solution to activate the free radical reaction, and the reaction time is 30 s-5 min.

[0011] (6) Quenching of labeling reaction: The quenching solution is a buffered saline solution containing 1-50 mM sodium ascorbate, 1-50 mM Trolox, and 1-50 mM sodium azide. Add it to the cells and let it stand for 30 s to 5 min. Repeat once and discard the quenching solution.

[0012] (7) Cell washing: Use one or more of the following: ammonium bicarbonate buffer solution, phosphate buffer solution, 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, or tris buffer solution at a pH of 7-10, and repeat the washing three times;

[0013] (8) Scrape or digest the cells with trypsin, disrupt the cells with ultrasound assistance or freeze-thaw with liquid nitrogen, at a temperature of 4-10°C for 10 min-2 h or 0-10°C for 10 min-10 h; centrifuge at 5000g-20000g for 5 min-30 min, and take the supernatant;

[0014] (9) adding an enrichment material prepared from an organic / inorganic material such as agarose gel beads, silica spheres, polymer spheres, etc., with one or more of streptavidin, avidin, etc. bonded to the surface to the protein variant solution extracted from the cells, and incubating with rotation at 4°C-30°C for 2h-24h;

[0015] (10) Nonspecific adsorption cleaning on the enrichment material: use one or more of 1%-5% sodium dodecyl sulfate, 1%-5% Triton X-100, 1%-5% Tween, 1%-5% NP-40, 0.1-8M sodium chloride solution, 0.1-8M potassium chloride solution, 0.1-1M sodium carbonate solution, 0.1-1M ammonium bicarbonate solution, and rotate and clean for 5 min-30 min at 4°C-30°C, and repeat the cleaning 1-5 times;

[0016] (11) Release of target protein variant complex: cleavage of sulfonyl-amide bond by adjusting pH to 4-10, temperature to 4°C-30°C, and reaction time to 2h-16h; release of enriched protein variant from enriched material and desalting;

[0017] (12) Accurate identification of target protein variant complexes: Protein variants are analyzed through a "top-down" strategy, using one of the following modes: high-energy collision dissociation (HCD), electron transfer fragmentation (ETD), electron transfer / high-energy collision dissociation (EThcD) fragmentation, and step-wise collision energy HCD (sceHCD) fragmentation, to increase the fragmentation efficiency of protein variants and achieve accurate identification of protein variants.

[0018] 1) The present invention can achieve specific labeling, enrichment and controlled release of target protein variant complexes through a proximity labeling probe containing a controllable cleavage group and an enrichment group; 2) Based on the proximity labeling-controlled release technology and the top-down mass spectrometry strategy, the information of the target protein variant and its interacting protein variants can be obtained, and the accurate identification of the complex can be achieved at the protein variant level.

[0019] The present invention has the following advantages:

[0020] 1. High labeling efficiency and rapid response: labeling can be achieved within 1 minute, with a small labeling radius and good complex labeling specificity;

[0021] 2. The cleavable group in the probe achieves the specific and efficient release of the labeled protein variant complex, and after cleavage and release, the tyrosine residue in the protein variant sequence introduces a positively charged amino group, which is beneficial to its mass spectrometry fragmentation and improves the accuracy of protein variant identification;

[0022] 3. Analyzing the complex at the protein variant level helps to accurately study the relevant molecular mechanisms of the target protein variant complex and explain the molecular mechanisms of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a short-range labeling probe structure containing a controllable cleavage group and an enrichment group;

[0024] Figure 2 It is a protein variant complex analysis technology based on proximity labeling-controlled release technology and "top-down" mass spectrometry strategy;

[0025] Figure 3 To characterize the membrane permeability of BSOP and BSSP probes;

[0026] Figure 4 Characterization of proteins labeled with RNase A, a standard protein, and BSOP probes after RNase A cleavage of BSOP;

[0027] Figure 5 Identify MS1 for the RNase A protein released after BSOP labeling;

[0028] Figure 6 Results of identification of mitochondrial protein variants in 293T cells;

[0029] Figure 7 Identification of coverage positions for protein variants in the mitochondrial electron transport and oxidative phosphorylation related protein variant complexes in 293T cells. DETAILED DESCRIPTION

[0030] Example 1: Synthesis of cleavable specific enrichment probe BSOP

[0031] (1) 0.391 g (1.6 mmol) of Biotin, 0.392 g (1.9 mmol) of DCC (dicyclohexylcarbodiimide), 0.028 g of DMAP (4-dimethylaminopyridine), and 0.76 g of 2,2-sulfonyldiethanol were dissolved in 12 mL of dimethyl sulfoxide (DMSO), and the solution was stirred at room temperature for 36 h. The mixture was purified by semi-preparative liquid chromatography (Hanbang Technology, equipped with a UV-visible 2000 detector, and the separation column was equipped with a C18 filler (10 μm, 50mm id×250mm)), with 50% volume concentration of acetonitrile as the mobile phase for isocratic elution for 30 min, the flow rate is 50 ml / min, the retention time is 22±1 min, and the product 1 (i.e. compound 1) is separated and purified as a white solid with a yield of 36%.

[0032] (2) Compound 1 was dissolved in 2 ml of DMSO (0.100 g, 0.262 mmol), N,N'-disuccinimidyl carbonate (0.101 g, 0.394 mmol) and 0.08 ml of pyridine (0.262 mmol) were added, and the mixture was stirred at room temperature for 6 h. The mixture was purified by semi-preparative liquid chromatography (Hanbang Technology, equipped with UV-visible 2000 detector, the separation column was equipped with C18 filler (10 μm, 50mm id×250mm), with a volume concentration of 20-40% acetonitrile linear gradient elution for 30min (collect by peak), a flow rate of 50ml / min, a retention time of 27±1min, and the separated and purified product 2 (i.e., compound 2) was in the form of a white solid with a yield of 40%;

[0033] (3) Compound 2 was dissolved in 1 ml of DMSO (0.100 g, 0.192 mmol), and tyramine (0.079 g, 0.576 mmol) was added. After fully dissolved, the mixture was stirred at room temperature for 30 min. Semi-preparative liquid chromatography (Hanbang Technology, equipped with UV-visible 2000 detector, separation column equipped with C18 filler (10 μm, 50mm id×250mm)), with 50% volume concentration of acetonitrile as the mobile phase for isocratic elution for 30min, flow rate of 50ml / min, retention time of 23±1min, the purified compound BSOP was isolated as a white solid with a yield of 90%; the compound structure was characterized by mass spectrometry, nuclear magnetic resonance hydrogen spectrum and carbon spectrum.

[0034]

[0035] Example 2: Synthesis of cleavable specific enrichment probe BSOPM

[0036] (1) Biotin 0.391 g (1.6 mmol), DCC (dicyclohexylcarbodiimide) 0.392 g (1.9 mmol), DMAP (4-dimethylaminopyridine) 0.028 g, 2,2-sulfonyldiethanol 0.76 g were dissolved in 12 mL of dimethyl sulfoxide (DMSO), and the solution was stirred at room temperature for 36 h. The mixture was purified by semi-preparative liquid chromatography (Hanbang Technology, equipped with UV-visible 2000 detector, the separation column was equipped with C18 filler (10 μm, 50mm id×250mm)), with 50% volume concentration of acetonitrile as the mobile phase, isocratic elution for 30 min, flow rate 50 ml / min, retention time 19±1 min, elution separation and purification of product 1 (i.e. compound 1), in the form of a white solid, with a yield of 36%.

[0037] (2) Compound 1 was dissolved in 2 ml DMSO (0.100 g, 0.262 mmol), N,N'-disuccinimidyl carbonate (0.101 g, 0.394 mmol) and 0.08 ml pyridine (0.262 mmol) were added, and stirred at room temperature for 6 h. Semi-preparative liquid chromatography (Hanbang Technology, equipped with UV-visible 2000 detector, separation column equipped with C18 filler (10 μm, 50mm id×250mm)), with a volume concentration of 20-40% acetonitrile gradient elution for 30min, a flow rate of 50ml / min, and a retention time of 24±1min, the product 2 (i.e., compound 2) was separated and purified in the form of a white solid with a yield of 40%.

[0038] (3) Compound 2 was dissolved in 1 ml of DMSO (0.100 g, 0.192 mmol), and 4-(2-aminoethyl)-2-methoxyphenol (0.096 g, 0.576 mmol) was added. After fully dissolved, the mixture was stirred at room temperature for 30 min. Semi-preparative liquid chromatography (Hanbang Technology, equipped with UV-visible 2000 detector, separation column equipped with C18 filler (10 μm, 50mm id×250mm)), with 50% volume concentration of acetonitrile as the mobile phase for isocratic elution for 30 min, the flow rate is 50 ml / min, the retention time is 28±1 min, the compound BSOPM is separated and purified as a white solid with a yield of 93%. The structure of the compound was characterized by mass spectrometry, nuclear magnetic resonance hydrogen spectrum and carbon spectrum.

[0039]

[0040] Example 3: Accurate identification of mitochondrial protein variant complexes in 293T cells

[0041] (1) A lentiviral recombinant plasmid carrying the mito-V5-APEX2 gene was constructed (Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY. (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 12, 51-4.), and 293T cells stably expressing a fusion protein of an N-terminal fusion mitochondrial matrix localization peptide, a Flag tag, and APEX2 were constructed (Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY. (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 12, 51-4.), and cultured in a 15 cm inner diameter culture dish with a confluence of 80-90% (a layer of cells covering 80-90% of the surface area of ​​the culture dish was observed under a microscope).

[0042] (2) Prepare 500 mM BSOP probe with DMSO, dilute to 0.5 mM with DMEM cell culture medium, add 10 mL to the cells in step (1), and incubate at 37° C. in an atmosphere of 5% carbon dioxide by volume for 30 min;

[0043] (3) Add 100 mM hydrogen peroxide solution at a volume ratio of 100:1 to the incubated cell culture medium, mix well, and let stand for 1 min;

[0044] (4) Prepare a buffered saline solution consisting of 10 mM sodium ascorbate, 5 mM Trolox, and 10 mM sodium azide as a quenching solution, mix well, add 10 ml of the solution to the cells, let stand for 2 min, repeat (add the quenching solution to the cells and let stand) once; discard the quenching solution;

[0045] (5) Add 6 ml of phosphate buffered saline (pH 7.4) and 10 ml of solution to wash the cells. Repeat the washing three times.

[0046] (6) Scrape the cells, add 1 ml of NP-40 lysis buffer (NP-40 lysis buffer, purchased from Biyuntian, China), and disrupt the cells with ultrasound at 4°C, 100 W (On 5 s, Off 5 s); centrifuge at 15000 g for 5 min, and collect the supernatant;

[0047] (7) Add 300 μl of Streptavidin Agarose Resin (purchased from Thermo Fisher Scientific (Rockford, USA)) to the protein variant solution extracted from the cells, and rotate at 4°C for 10 hours to enrich the probe-labeled protein variants; centrifuge at 1700 g, remove the supernatant, and store the Streptavidin Agarose Resin enriched with the probe-labeled protein variants at 4°C.

[0048] (8) Elution of protein variants nonspecifically adsorbed on the enrichment material: Use 1 ml of NP-40 lysis buffer (NP-40 lysis buffer, purchased from Biyuntian, China), rotate and wash at 4°C for 5 min, repeat the washing three times; then use 1 ml of phosphate buffered saline, rotate and wash at 4°C for 5 min, repeat the washing three times;

[0049] (9) Protein variant release: 1 M ammonium bicarbonate solution (pH 8.0) was added to release the enriched protein variants from the enrichment material, and liquid phase offline desalting was performed using a C1-trap column (inner diameter 75 μM, length 10 cm); a sample of the protein variant composition of the mitochondrial protein variant complex was obtained.

[0050] (10) The protein variant samples were analyzed using top-down mass spectrometry (Toby TK, Fornelli L, Kelleher NL. (2016) Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu Rev Anal Chem (Palo Alto Calif). 9, 499-519.), using an Orbitrap Fusion Lumos mass spectrometer in sceHCD mass spectrometry acquisition mode.

[0051] (11) The collected protein variant data were searched using TopPIC software, and the database was downloaded from the Uniprot website (Homo sapiens human complete proteome), and 437 protein variants were accurately identified, including P25705_44_75, P25705_44_82, P25705_44_83, P25705_44_85, P25705_57_85, P25705_51_85, P25705_58_85, P25705_86_133, P25705_44_88, P25705_57_83 variants corresponding to the P25705 protein. Example 4: Accurate identification of protein variants of the α-synuclein complex

[0052] (1) A lentiviral recombinant plasmid carrying the SNCA-APEX2 gene was constructed (Chung CY, Khurana V, Yi S, Sahni N, Loh KH, Auluck PK, Baru V, Udeshi ND, Freyzon Y, Carr SA, Hill DE, Vidal M, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell Syst. 4, 242-250.), and the lentivirus was successfully transduced into the target SH-SY5Y cells, successfully constructing SH-SY5Y cells stably expressing the α-synuclein-APEX2 fusion protein (Chung CY, Khurana V, Yi S, Sahni N, Loh KH, Auluck PK, Baru V, Udeshi ND, Freyzon Y, Carr SA, Hill DE, Vidal M, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell Syst. 4, 242-250.). SA, Hill DE, Vidal M, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell Syst. 4, 242-250.), cultured to 80-90% confluence (a layer of cells covering 80-90% of the surface area of ​​the culture dish was observed under a microscope).

[0053] (2) Prepare 500 mM BSOPM probe with DMSO, dilute to 0.5 mM with DMEM cell culture medium, add 10 ml to the cells in step (1), and incubate at 37° C. in an atmosphere of 5% carbon dioxide by volume for 30 min;

[0054] (3) Add 100 mM hydrogen peroxide solution at a volume ratio of 100:1 to the incubated cell culture medium, mix well, and let stand for 1 min;

[0055] (4) Prepare a buffered saline solution consisting of 10 mM sodium ascorbate, 5 mM Trolox, and 10 mM sodium azide as a quenching solution, mix well, add 10 ml of the solution to the cells, let stand for 2 min, repeat (add the quenching solution to the cells and let stand) once; discard the quenching solution;

[0056] (5) Add 6 ml of phosphate buffered saline (pH 7.4) and 10 ml of solution to wash the cells. Repeat the washing three times.

[0057] (6) Scrape the cells, add 1 ml of NP-40 lysis buffer (NP-40 lysis buffer, purchased from Biyuntian, China), and disrupt the cells with ultrasound at 4°C, 100 W (On 5 s, Off 5 s); centrifuge at 15000 g for 5 min, and collect the supernatant;

[0058] (7) Add 300 μl of Streptavidin Agarose Resin (purchased from Thermo Fisher Scientific (Rockford, USA)) to the protein variant solution extracted from the cells, and rotate at 4°C for 10 hours to enrich the probe-labeled protein variants; centrifuge at 1700 g, remove the supernatant, and store the Streptavidin Agarose Resin enriched with α-synuclein and its interacting protein variants at 4°C.

[0059] (8) Elution of protein variants nonspecifically adsorbed on the enrichment material: Use 1 ml of NP-40 lysis buffer (NP-40 lysis buffer, purchased from Biyuntian, China), rotate and wash at 4°C for 5 min, repeat the washing three times; then use 1 ml of phosphate buffered saline, rotate and wash at 4°C for 5 min, repeat the washing three times;

[0060] (9) Protein variant release: 1 M ammonium bicarbonate solution (pH 8.0) was added to release the enriched protein variants from the enrichment material, and liquid phase offline desalting was performed using a C1-trap column (inner diameter 75 μM, length 10 cm); samples of α-synuclein and its interacting protein variants were obtained.

[0061] (10) The protein variant samples were analyzed using top-down mass spectrometry (Toby TK, Fornelli L, Kelleher NL. (2016) Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu Rev Anal Chem (Palo Alto Calif). 9, 499-519.), using an Orbitrap Fusion Lumos mass spectrometer in HCD mass spectrometry acquisition mode.

[0062] (11) The collected protein variant data were searched using TopPIC software, and 127 variants of α-synuclein and its interacting proteins were accurately identified, including the P37840_44_140, P37840_44_126, P37840_40_140, P37840_57_140, and P37840_51_140 variants corresponding to the P37840 protein.

[0063] Example 5: Accurate identification of protein variants in the α-synuclein complex

[0064] (1) A lentiviral recombinant plasmid carrying the SNCA-APEX2 gene was constructed (Chung CY, Khurana V, Yi S, Sahni N, Loh KH, Auluck PK, Baru V, Udeshi ND, Freyzon Y, Carr SA, Hill DE, Vidal M, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell Syst. 4, 242-250.), and the lentivirus was successfully transduced into the target SH-SY5Y cells, successfully constructing SH-SY5Y cells stably expressing the α-synuclein-APEX2 fusion protein (Chung CY, Khurana V, Yi S, Sahni N, Loh KH, Auluck PK, Baru V, Udeshi ND, Freyzon Y, Carr SA, Hill DE, Vidal M, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell Syst. 4, 242-250.). SA, Hill DE, Vidal M, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell Syst. 4, 242-250.), cultured to 80-90% confluence (a layer of cells covering 80-90% of the surface area of ​​the culture dish was observed under a microscope).

[0065] (2) Prepare 500 mM BSOP probe with DMSO, dilute to 0.5 mM with DMEM cell culture medium, add 10 ml to the cells in step (1), and incubate at 37° C. in an atmosphere of 5% carbon dioxide by volume for 30 min;

[0066] (3) Add 100 mM hydrogen peroxide solution at a volume ratio of 100:1 to the incubated cell culture medium, mix well, and let stand for 1 min;

[0067] (4) Prepare a buffered saline solution consisting of 10 mM sodium ascorbate, 5 mM Trolox, and 10 mM sodium azide as a quenching solution, mix well, add 10 ml of the solution to the cells, let stand for 2 min, repeat (add the quenching solution to the cells and let stand) once; discard the quenching solution;

[0068] (5) Add 6 ml of phosphate buffered saline (pH 7.4) and 10 ml of solution to wash the cells. Repeat the washing three times.

[0069] (6) Scrape the cells, add 1 ml of NP-40 lysis buffer (NP-40 lysis buffer, purchased from Biyuntian, China), and disrupt the cells with ultrasound at 4°C, 100 W (On 5 s, Off 5 s); centrifuge at 15000 g for 5 min, and collect the supernatant;

[0070] (7) Add 300 μl of Streptavidin Agarose Resin (purchased from Thermo Fisher Scientific (Rockford, USA)) to the protein variant solution extracted from the cells, and rotate at 4°C for 10 hours to enrich the probe-labeled protein variants; centrifuge at 1700 g, remove the supernatant, and store the Streptavidin Agarose Resin enriched with α-synuclein and its interacting protein variants at 4°C.

[0071] (8) Elution of protein variants nonspecifically adsorbed on the enrichment material: Use 1 ml of NP-40 lysis buffer (NP-40 lysis buffer, purchased from Biyuntian, China), rotate and wash at 4°C for 5 min, repeat the washing three times; then use 1 mL of phosphate buffered saline, rotate and wash at 4°C for 5 min, repeat the washing three times;

[0072] (9) Protein variant release: A 50 mM ammonium bicarbonate solution with a final concentration of 10 mM DTT was added to the washed streptavidin agarose beads to release the enriched protein variants from the enriched material. The beads were then desalted offline using a C1-trap column (inner diameter 75 μM, length 10 cm) to obtain samples of α-synuclein and its interacting protein variants.

[0073] (10) The protein variant samples were analyzed using top-down mass spectrometry (Toby TK, Fornelli L, Kelleher NL. (2016) Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu Rev Anal Chem (Palo Alto Calif). 9, 499-519.), using an Orbitrap Fusion Lumos mass spectrometer in sceHCD mass spectrometry acquisition mode.

[0074] (11) The collected protein variant data were searched using TopPIC software, and the database was downloaded from the Uniprot website (Homo sapiens human complete proteome), and 437 protein variants were accurately identified, including P25705_44_75, P25705_44_82, P25705_44_83, P25705_44_85, P25705_57_85, P25705_51_85, P25705_58_85, P25705_86_133, P25705_44_88, P25705_57_83 variants corresponding to the P25705 protein. Example 6: Accurate identification of protein variants of the α-synuclein complex in subcellular organelles

[0075] (1) Construction of lentiviral recombinant plasmid carrying mito-V5-SNCA-APEX2 gene (Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY. (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 12, 51-4. Chung CY, Khurana V, Yi S, Sahni N, Loh KH, Auluck PK, Baru V, Udeshi ND, Freyzon Y, Carr SA, Hill DE, Vidal M, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell Syst.4,242-250.), and the lentivirus was successfully transduced into the target cell SH-SY5Y cells, and the SH-SY5Y cells with N-terminal fusion of mitochondrial matrix localization peptide and C-terminal fusion of α-synuclein horseradish peroxidase were constructed (Lam SS, Martell JD, Kamer KJ, Deerinck TJ, EllismanMH, Mootha VK, Ting AY. (2015) Directed evolution of APEX2 for electronmicroscopy and proximity labeling. Nat Methods.12,51-4. Chung CY, Khurana V, YiS, Sahni N, Loh KH, Auluck PK, Baru V, Udeshi ND, Freyzon Y, Carr SA, Hill DE, VidalM, Ting AY, Lindquist S. (2017) In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons.Cell Syst.4, 242-250.), cultured to 80-90% confluence (a layer of cells covering 80-90% of the surface area of ​​the culture vessel was observed under a microscope).

[0076] (2) Prepare 500 mM BSOPM probe with DMSO, dilute to 0.5 mM with DMEM cell culture medium, add 10 ml to the cells in step (1), and incubate at 37° C. in an atmosphere of 5% carbon dioxide by volume for 30 min;

[0077] (3) Add 100 mM hydrogen peroxide solution at a volume ratio of 100:1 to the incubated cell culture medium, mix well, and let stand for 1 min;

[0078] (4) Prepare a buffered saline solution consisting of 10 mM sodium ascorbate, 5 mM Trolox, and 10 mM sodium azide as a quenching solution, mix well, add 10 ml of the solution to the cells, let stand for 2 min, repeat (add the quenching solution to the cells and let stand) once; discard the quenching solution;

[0079] (5) Add 6 ml of phosphate buffered saline (pH 7.4) and 10 ml of solution to wash the cells. Repeat the washing three times.

[0080] (6) Scrape the cells, add 1 ml of NP-40 lysis buffer (NP-40 lysis buffer, purchased from Biyuntian, China), and disrupt the cells with ultrasound at 4°C, 100 W (On 5 s, Off 5 s); centrifuge at 15000 g for 5 min, and collect the supernatant;

[0081] (7) Add 300 μl of Streptavidin Agarose Resin (purchased from Thermo Fisher Scientific (Rockford, USA)) to the protein variant solution extracted from the cells, and rotate at 4°C for 10 hours to enrich the probe-labeled protein variants; centrifuge at 1700 g, remove the supernatant, and store the Streptavidin Agarose Resin enriched for α-synuclein and its interacting protein variants in mitochondria at 4°C.

[0082] (8) Elution of protein variants nonspecifically adsorbed on the enrichment material: Use 1 mL of 1% sodium dodecyl sulfate, rotate and wash at 4°C for 5 min, repeat the washing three times; then use 1 mL of phosphate buffered saline, rotate and wash at 4°C for 5 min, repeat the washing three times; then use 1 mL of 3M sodium chloride, rotate and wash at 4°C for 5 min, repeat the washing three times;

[0083] (9) Protein variant release: 1 M ammonium bicarbonate solution (pH 8.0) was added to release the enriched protein variants from the enrichment material, and liquid phase offline desalting was performed using a C1-trap column (inner diameter 75 μM, length 10 cm); a sample of the protein variant composition of the mitochondrial protein variant complex was obtained.

[0084] (10) The protein variant samples were analyzed using top-down mass spectrometry technology (Toby TK, Fornelli L, Kelleher NL. (2016) Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu Rev Anal Chem (Palo Alto Calif). 9, 499-519.) using an Orbitrap Fusion Lumos mass spectrometer and the EThcD fragmentation mode.

[0085] (11) The collected protein variant data were searched using TopPIC software, and 127 variants of α-synuclein and its interacting proteins were accurately identified, including the P37840_44_140, P37840_44_126, P37840_40_140, P37840_57_140, and P37840_51_140 variants corresponding to the P37840 protein.

Claims

1. A method for analyzing protein variant complexes based on proximity labeling-controlled release, characterized in that: A proximity labeling probe containing controllable cleavage groups and enrichment groups is used to perform in situ proximity labeling, enrichment and controllable release of the target protein variant complex, and a "top-down" mass spectrometry strategy is further used to accurately identify the target protein variants and their interacting protein variants.

2. The analysis method according to claim 1, characterized in that: The specific process is as follows: 1) constructing a cell line expressing a fusion protein of peroxidase and target protein; using a probe containing a controllable cleavage group and an enrichment group to label the target protein variant complex in a short distance: adding a cell culture solution containing the probe to one or more of bacteria, fungi or cells for incubation, adding a hydrogen peroxide solution for free radical reaction activation, and then adding a quenching solution; 2) washing one or more of the bacteria, fungi or cells obtained in step 1) and then disrupting the cells; Then, a material that reacts with the enrichment group in the probe is added to the cell lysate to enrich the labeled target protein variant complex; 3) washing away nonspecifically adsorbed protein variants on the enrichment material using surfactants and / or salt solutions; 4) Using the cleavable group on the probe, the probe can be controlled to release the target protein and its interacting protein variants; 5) Use a "top-down" mass spectrometry strategy to accurately identify the released target protein variants and their interacting protein variants.

3. The analysis method according to claim 1 or 2, characterized in that: The short-range labeling probe is in the form of a chain, one end of which is a phenol group that can be used for short-range labeling, the other end of which is a biotin group and / or an alkyne group that can be used for specific enrichment, and the middle of which has a controllable cleavage group, one of the sulfonyl-amide bonds; The short-range labeling probe specifically includes one or more of BSOP and BSOPM.

4. The analysis method according to claim 1 or 2, characterized in that: The protein variant complex is a mixture of protein variants composed of interacting protein variants; The bacteria, fungi or cells are one of the cell lines in which the peroxidase and the target protein are fused by a cloning construction method.

5. The analysis method according to claim 2, characterized in that: The process of short-range labeling of the target protein variant complex with the probe containing a controllable cleavage group and an enrichment group in step (1) is as follows: dissolving the probe containing a controllable cleavage group and an enrichment group in dimethyl sulfoxide and / or dimethylformamide, adding cell culture medium to dilute the probe concentration to a range of 1 μM to 1 M, and then using the culture medium to add one or more of bacteria, fungi or cells for incubation at a temperature of 36-38° C. and an air volume concentration of 2-10% carbon dioxide for a time of 30 min to 2 h; adding a hydrogen peroxide solution with a concentration of 0.5-100 mM for free radical reaction activation for a reaction time of 30 s to 5 min, and then adding a quenching solution, the quenching solution being a buffered salt solution containing 1-50 mM sodium ascorbate, 1-50 mM Trolox, and 1-50 mM sodium azide for a quenching time of 30 s to 5 min.

6. The analysis method according to claim 2, characterized in that: The specific enrichment of the labeled protein variants described in step (2) is achieved by specific binding between the biotin on the probe and the specific enrichment material, wherein the specific enrichment material includes an enrichment material prepared from one or more organic / inorganic materials such as agarose gel beads, silica balls, polymer balls, etc., with monomers such as one or more of streptavidin, avidin, etc. The enrichment temperature is 4°C to 30°C, and the enrichment time is 2h-48h.

7. The analysis method according to claim 2, characterized in that: The cleaning solution in step (3) is one or more of the following: sodium dodecyl sulfate solution with a mass concentration of 1%-5%, Triton X-100 solution with a mass concentration of 1%-5%, Tween solution with a mass concentration of 1%-5%, NP-40 solution with a mass concentration of 1%-5%, sodium chloride solution with a mass concentration of 0.1-8M, potassium chloride solution with a mass concentration of 0.1-8M, sodium carbonate solution with a mass concentration of 0.1-1M, and ammonium bicarbonate solution with a mass concentration of 0.1-1M.

8. The analysis method according to claim 2, characterized in that: The controllable cleavage probe method of step (4), such as when the controllable cleavage group is a disulfide bond, uses 10-100mM dithiothreitol (DTT) and or tri-(2-carboxyethyl)phosphine hydrochloride (TCEP), the temperature is 25-95°C, and the reaction time is 30min-120min; when the controllable cleavage group is a sulfonyl-amide bond, the pH is adjusted to 4-10, the temperature is 4°C-30°C, and the reaction time is 2h-16h.

9. The analysis method according to claim 2, characterized in that: Step (5) is achieved by top-down mass spectrometry technology: the released target protein and its interacting protein variants are separated by liquid chromatography and then ionized and fragmented in a mass spectrometer, thereby accurately identifying the complete amino acid sequence and post-translational modification sites of the protein variants; the fragmentation mode includes one or more of high energy collision dissociation (HCD), electron transfer fragmentation (ETD), and electron transfer / high energy collision dissociation (EThcD) fragmentation modes.

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