Proximity protein marking method and application

Abstract

The invention discloses an adjacent protein marking method and application. The invention provides an adjacent protein labeling method, which comprises the following steps: by taking an aldehyde / keto-phenol small-molecule probe as a substrate, catalyzing the substrate into a substrate containing active free radicals by using a catalyst, and forming a covalent label by the substrate containing the active free radicals and a target protein to obtain a labeled protein. According to the method, the aldehyde / keto-phenol probe with high reaction activity is combined with a hydrazide / hydroxylamine reversible enrichment process, so that marker site identification with high sensitivity and high coverage is realized, and the limitations of site level analysis, cell physiological disturbance and the like of a traditional proximity labeling method are overcome. The method is simple to operate, good in reproducibility, high in data result reliability and accuracy and more real and reliable in identification result. By means of efficient site level analysis, the proteome can be identified and marked more accurately, and more sensitive protein structure analysis is realized.

Description

Technical Field

[0001] This invention relates to the field of protein analysis technology, and more specifically, to a method and application of neighboring protein labeling. Background Technology

[0002] Deciphering the spatial distribution and interaction networks of subcellular proteomes is crucial for understanding their precise biological functions. In recent years, enzyme-mediated proximity labeling (PL) has become an effective tool for mapping subcellular proteomes and studying protein-protein interaction networks. These methods target engineered enzymes (APEX2, BioID, and TurboID) genes to specific subcellular compartments or fuse them with target proteins for expression, catalyzing small molecule probes to generate bioactive molecular intermediates, thereby achieving covalent labeling of neighboring proteins at the nanoscale.

[0003] Compared to traditional methods (biochemical fractionation and immunoprecipitation), proximity labeling (PL) offers significant advantages: This technique is performed within living cells, eliminating the need for prior cell lysis, thus enabling the capture of low-affinity and transient protein interactions with high spatiotemporal resolution. Furthermore, it eliminates the need for organelle purification, avoiding the contamination problems associated with biochemical fractionation. Notably, this technique can also capture proteomic information from non-purifiable subcellular regions (such as the synaptic cleft), which is impossible with traditional methods.

[0004] However, current proximity labeling techniques still face significant challenges, including limitations imposed by site-level analysis and cellular physiological perturbations. For example, traditional proximity labeling techniques primarily focus on protein-level analysis (Protein ID), but the binding of streptavidin-beads (SA-beads) to non-specific proteins increases the false positive rate and affects data accuracy. This is because traditional proximity labeling techniques typically rely on biotin probe labeling and streptavidin enrichment processes, but the irreversible binding of biotin to streptavidin leads to suboptimal recovery rates of labeled peptides, making site-level analysis of labeled proteins difficult. Larger biotinylated structures also generate more characteristic fragment ions, complicating spectral interpretation and further impacting site identification efficiency. On the other hand, engineered enzymes may cause potential cellular physiological perturbations during application. For instance, biotin ligases (BioID and TurboID) inevitably lead to widespread biotinylation of endogenous proteins, affecting cellular physiological processes. Peroxidase (APEX / APEX2) labeling typically requires a high concentration (1 mM) of H2O2 to initiate the reaction, which can cause cellular oxidative toxicity and perturbation of endogenous protein post-translational modifications, severely affecting the reliability of results from oxidatively sensitive disease models.

[0005] Several peroxidase (APEX2) labeling strategies have been developed to improve site recognition efficiency, such as alternative labeling probes (alkynyl-phenol and dethiobiotin-phenol) and optimized enrichment procedures. Alynyl-phenol (Alk-Ph) is a click-chemical peroxidase substrate that has been successfully used to label yeast cell mitochondrial matrix. Alk-Ph-labeled peptides can be recovered by streptavidin enrichment and UV cleavage for resolving mitochondrial membrane protein topological maps. To more efficiently recover labeled peptides, researchers are also using dethiobiotin-phenol (DBP) as a probe, which effectively weakens the interaction between biotin and streptavidin and is used for topological analysis of the inner mitochondrial membrane (IMM) proteome. These site-specific methods not only directly identify labeled subcellular proteomes (reducing false positive rates) but also provide information on the accessibility of enzymatic labeling to protein structures, which helps in deducing subcellular membrane protein topology and resolving dynamic conformational changes in proteins.

[0006] However, although these methods improve site identification efficiency, they ignore the physiological disturbance of cells caused by high concentrations (1mM) H2O2 when applied, and can only solve some of the limitations of traditional proximity labeling, thus limiting the development and application of proximity protein labeling technology. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a method and application for neighboring protein labeling.

[0008] To overcome these challenges, this invention provides an aldehyde / ketone-phenol probe-based proximity labeling technology (Ald-PL / Ket-PL), a novel proximity labeling strategy that combines highly reactive aldehyde / ketone-phenol probes with a chemically reversible acylhydrazine / hydroxylamine enrichment process for peroxidase or photocatalytic labeling, achieving highly sensitive labeling site identification under low oxidation perturbation. This invention innovates in probe optimization (higher labeling efficiency), enrichment process (reversible enrichment process), and mass spectrometry compatibility (mass spectrometry detection sensitivity), significantly improving site identification efficiency. In the peroxidase-protein system, the high reactivity of the aldehyde / ketone-phenol probe allows the labeling reaction to be efficiently initiated at extremely low H2O2 concentrations (100 μM). Compared to traditional peroxidase labeling methods, the amount of H2O2 required is reduced by 10 times. In photocatalyst-protein systems, aldehyde / keto-phenol probes can achieve efficient labeling under visible light irradiation (blue light, 420-430 nm), avoiding the introduction of H2O2. This greatly reduces or avoids H2O2-induced cytotoxicity and perturbation of endogenous post-translational modifications (PTMs), making Ald-PL / Ket-PL particularly promising for accurately mapping subcellular PTMs in redox-sensitive biological systems and under physiological conditions. Furthermore, Ald-PL / Ket-PL has a wide range of applications and prospects, enabling efficient catalytic labeling (peroxidases or photocatalysts) in living cells or in vitro under near-physiological conditions (low H2O2 concentration or no H2O2). Ald-PL / Ket-PL can effectively label proteins under non-denaturing conditions in vitro, achieving highly sensitive and high-coverage labeling site identification, offering significant advantages in structural biology, drug target discovery, and protein-protein interaction network research. In live-cell systems, Ald-PL / Ket-PL not only allows for more accurate identification of labeled proteomes using labeled sites without the need for control groups, but also enables comprehensive mapping of subcellular membrane protein topology and highly sensitive monitoring of dynamic protein conformational changes. This technology represents a significant advancement in the field of proximity protein labeling, overcoming limitations of traditional proximity labeling such as cellular physiological perturbations and site-level analysis. This novel proximity protein labeling system expands the application scope of proximity protein labeling technology.

[0009] To achieve the above objectives, the present invention is implemented through the following solution:

[0010] In a first aspect, the present invention provides a method for labeling adjacent proteins, using an aldehyde / keto-phenol small molecule probe as a substrate, catalyzing the substrate into a substrate containing active free radicals, wherein the substrate containing active free radicals forms a covalent label with the target protein to obtain a labeled protein;

[0011] The catalyst is a peroxidase or a photocatalyst;

[0012] The catalyst and / or target protein are located in a catalyst-protein system;

[0013] The catalyst-protein system includes an extracellular catalyst-protein system or an intracellular catalyst-protein system.

[0014] The catalyst-protein extracellular system is a mixture containing the catalyst and the target protein;

[0015] The catalyst-protein intracellular system is a cell containing a fusion protein of peroxidase and the target protein; or a cell containing a subcellular-targeting photocatalyst and the target protein.

[0016] Furthermore, the fusion protein includes a fusion protein containing a peroxidase and a subcellular localization signal peptide. The peroxidase can be targeted to specific subcellular regions (such as the mitochondrial matrix, endoplasmic reticulum, cell membrane, etc.), or it can be fused with a specific target protein for expression to study protein-protein interaction networks.

[0017] Furthermore, the subcellular targeted photocatalyst includes a photocatalyst conjugated with an antibody against the target protein or a photocatalyst that itself has subcellular localization.

[0018] Further, the aldehyde / ketone-phenol small molecule probe is an aromatic compound with a phenolic hydroxyl, aldehyde, or ketone group as its backbone structure, and its structure simultaneously contains phenolic hydroxyl, aldehyde, or ketone functional groups; preferably, the aldehyde / ketone-phenol small molecule probe includes at least one of salicylaldehyde (Sal), 3-hydroxybenzaldehyde (3-Hba), p-hydroxybenzaldehyde (p-Hba), 2,6-dimethyl-4-hydroxybenzaldehyde, and p-hydroxyacetophenone.

[0019] Furthermore, when the catalyst is peroxidase, H2O2 with a final concentration of 10~1000 μM is added to initiate peroxidase catalytic labeling, preferably with a final H2O2 concentration of 100 μM.

[0020] Furthermore, the peroxidase includes horseradish peroxidase or ascorbic acid peroxidase.

[0021] Furthermore, the labeling time is 1~10 min, and the labeling temperature is 25~37℃.

[0022] Furthermore, when the catalyst is peroxidase, the labeling time is 1-3 min and the labeling temperature is 25-37°C; preferably, the labeling time is 3 min and the labeling temperature is 37°C.

[0023] Furthermore, when the catalyst is a photocatalyst, the labeling time is 1~10 min and the labeling temperature is 25~37℃; preferably, the labeling time is 10 min and the labeling temperature is 25℃.

[0024] Furthermore, in the catalyst-protein extracellular system, when the catalyst is a peroxidase, the final concentration of the peroxidase is 0.1~0.5 mg / ml, preferably 0.5 mg / ml.

[0025] Furthermore, when the catalyst is a peroxidase, an aldehyde / keto-phenol small molecule probe is first added to the catalyst-protein system for incubation, and then H2O2 is added to initiate the peroxidase catalytic labeling.

[0026] Furthermore, when the catalyst is a photocatalyst, the photocatalytic labeling is initiated using visible light with a wavelength of 380~780 nm. Preferably, the visible light wavelength is 420~430 nm.

[0027] Furthermore, the photocatalyst includes an organic small molecule photocatalyst or a transition metal photocatalyst;

[0028] Preferably, the organic small molecule photocatalyst includes riboflavin and / or eosin Y, and the transition metal photocatalyst includes a ruthenium catalyst.

[0029] Furthermore, the final concentration of the photocatalyst is 1~20 μM. Preferably, the final concentration of the catalyst is 10 μM.

[0030] Furthermore, when the catalyst is a photocatalyst, an aldehyde / keto-phenol small molecule probe is first added to the catalyst-protein system for incubation, and then the visible light is irradiated to initiate photocatalytic labeling.

[0031] Furthermore, when the catalyst-protein system is an extracellular catalyst-protein system, an aldehyde / keto-phenol small molecule probe is added and incubated for 1-5 min, preferably 2 min; when the catalyst-protein system is an intracellular catalyst-protein system, an aldehyde / keto-phenol small molecule probe is added and incubated for 20-60 min, preferably 30 min.

[0032] Furthermore, the concentration of the aldehyde / keto-phenol small molecule probe is 0.1~16 mM, preferably 4 mM.

[0033] Furthermore, when the catalyst is a peroxidase, the catalytic labeling reaction is terminated with a terminating reagent, which includes sodium azide, sodium ascorbate, and quinoline dimethacrylate; when the catalyst is a photocatalyst, visible light irradiation is stopped to terminate the catalytic labeling reaction.

[0034] Furthermore, when the catalyst-protein system is a peroxidase-protein extracellular system, a termination reagent (sodium azide, sodium ascorbate, and quinoline dimethacrylate) is added to the reaction system that initiates the peroxidase-catalyzed labeling reaction to terminate the catalytic labeling reaction; when the catalyst-protein system is a peroxidase-protein intracellular system, a termination solution is prepared by adding the termination reagent (sodium azide, sodium ascorbate, and quinoline dimethacrylate) to PBS buffer (10 mM). After the cells are labeled, the reaction solution is immediately discarded, and the termination solution is added to terminate the catalytic labeling reaction.

[0035] Furthermore, the working concentration of sodium azide, sodium ascorbate, and quinoline dimethacrylate in the terminating reagent is 10-20 mM.

[0036] Preferably, the working concentration of sodium azide, sodium ascorbate, and quinoline dimethacrylate in the terminating reagent is 10 mM.

[0037] Furthermore, when the catalyst is peroxidase, a terminating reagent is added to ice for 5-10 minutes to terminate the catalytic labeling reaction, preferably 10 minutes.

[0038] Furthermore, when the catalyst-protein system is a catalyst-protein extracellular system, cold acetone is added after terminating the catalytic labeling reaction, and the mixture is precipitated overnight at -30°C to obtain the labeled protein.

[0039] Furthermore, the volume ratio of the terminated solution to cold acetone after terminating the catalytic labeling reaction is 1:(4~6), preferably 1:5.

[0040] Furthermore, when the catalyst-protein system is an intracellular catalyst-protein system, the cells after the catalytic labeling reaction is terminated are labeled cells. After washing with PBS buffer, the labeled cells can be lysed to obtain the labeled protein.

[0041] Secondly, the present invention provides a method for protein level analysis, comprising the following steps:

[0042] S11: Construct a solid support for the coupled labeled protein, coupled the labeled protein obtained by the adjacent protein labeling method with a solid support containing specific groups, and collect the enzymatic hydrolysate after reduction, alkylation reaction and enzymatic hydrolysis.

[0043] S12: Acidify and desalt the enzymatic hydrolysate from step S11, and then perform direct mass spectrometry detection;

[0044] Or, S13: using Ti 4+ Immobilized metal affinity chromatography (IMAC) polymer microspheres were used to enrich and elute phosphopeptides in the enzymatic hydrolysate of step S11. The eluent was collected, desalted, or fractionated by high pH reversed-phase chromatography, and then detected by mass spectrometry.

[0045] The specific groups include hydrazide and / or hydroxylamine groups, and the solid support includes magnetic beads, resin or agarose.

[0046] Further, in step S11, the labeled protein solution is mixed with a solid support containing specific groups and subjected to a oscillating reaction to obtain a solid support for the coupled labeled protein; the solid support for the coupled labeled protein is mixed with urea, and dithiothreitol (DTT) is added for a oscillating reduction reaction, followed by the addition of iodoacetamide (IAA) for an oscillating alkylation reaction to obtain a solid support for the coupled labeled protein after reduction and alkylation reactions.

[0047] Further, in step S11, when the catalyst-protein system is an extracellular catalyst-protein system, the labeled protein solution is a labeled protein resuspension of the labeled protein resuspended in enrichment buffer 1; when the catalyst-protein system is an intracellular catalyst-protein system, the labeled cells are resuspended in enrichment buffer 1, and cell lysis is performed to obtain the labeled protein solution.

[0048] The enrichment buffer 1 is a citrate-sodium citrate buffer containing sodium dodecyl sulfate.

[0049] Furthermore, in step S11, the enrichment buffer 1 is a citrate-sodium citrate buffer containing 2-5% w / v sodium dodecyl sulfate, with a pH of 4.4-4.6; preferably, the concentration of sodium dodecyl sulfate is 4% w / v.

[0050] Furthermore, in step S11, the oscillation reaction temperature is 25~37℃ and the time is more than 20 h, preferably 37℃.

[0051] Furthermore, in step S11, when the catalyst is a peroxidase, the ratio of the labeled protein to the solid support with specific groups is 1~3 mg:(50~100) μL, and when the catalyst-protein system is a catalyst-protein extracellular system, it is preferably 1.5 mg:100 μL, and when the catalyst-protein system is a catalyst-protein intracellular system, it is preferably 3 mg:100 μL.

[0052] Furthermore, in step S11, when the catalyst is a photocatalyst, the ratio of the labeled protein to the solid support with specific groups is 0.1~3 mg: (10~100) μL, and when the catalyst-protein system is a catalyst-protein extracellular system, it is preferably 0.2 mg: 15 μL; when the catalyst-protein system is a catalyst-protein intracellular system, it is preferably 3 mg: 100 μL.

[0053] Furthermore, in step S11, the concentration of urea is 2~8M, preferably 8M.

[0054] Furthermore, in step S11, the final concentration of the dithiothreitol is 10-20 mM, preferably 10 mM; and the final concentration of the iodoacetamide is 20-40 mM, preferably 20 mM.

[0055] Furthermore, in step S11, the reduction reaction temperature is 37~56℃ and the time is 1~2 h, preferably 56℃ and the time is 1 h; the alkylation reaction is incubated at room temperature in the dark for 30~40 min, preferably 30 min.

[0056] Furthermore, in step S11, the solid support of the coupled labeled protein from the reduction and alkylation reactions is cleaned to remove non-specifically adsorbed proteins, trypsin is added for enzymatic digestion, and the on-bead digest is collected.

[0057] Furthermore, in step S11, the equipment is cleaned with urea, sodium chloride, and ammonium bicarbonate, respectively.

[0058] Furthermore, in step S11, the equipment is cleaned with 2-8 M urea, 1-2 M sodium chloride, and 20-100 mM ammonium bicarbonate, respectively; preferably 8 M urea, 1.5 M sodium chloride, and 100 mM ammonium bicarbonate.

[0059] Furthermore, in step S11, the mass ratio of trypsin to protein is 1:(20~50), preferably 1:25.

[0060] Furthermore, in step S11, trypsin is added and enzymatically hydrolyzed in 20-100 mM ammonium bicarbonate at 25-37°C for at least 16 hours on a shaker, with a hydrolysis volume of 400-600 μL; preferably, enzymatically hydrolyzed in 100 mM ammonium bicarbonate at 37°C on a shaker for at least 16 hours, with a hydrolysis volume of 600 μL.

[0061] Furthermore, in step S13, the enzymatic hydrolysate from step S11 is mixed with the enrichment solution, and Ti is added. 4+ -IMAC microspheres are reacted on an oscillator at 25~37℃ for 0.5~3 h to enrich phosphopeptides; the enrichment solution contains acetonitrile and formic acid; preferably, the reaction is carried out at 25℃ for 2 h.

[0062] Furthermore, in step S13, the enrichment solution contains 70-80% v / v acetonitrile and 5-6% v / v formic acid, preferably 80% v / v acetonitrile and 6% v / v formic acid.

[0063] Furthermore, in step S13, the volume ratio of the enzymatic hydrolysate to the enrichment solution is 1:(0.9~1.2), preferably 1:1.

[0064] Furthermore, in step S13, the enzymatic hydrolysate and Ti 4+ The ratio of -IMAC microspheres is 500 μL: 5~10 mg, preferably 500 μL: 10 mg.

[0065] Further, in step S13, the washing solution is used to wash the food at 25-37°C for 30-40 min on a shaker with washing solution 1 and washing solution 2 respectively; washing solution 1 contains acetonitrile, sodium chloride and formic acid; washing solution 2 contains acetonitrile and formic acid; preferably, the washing is carried out at 25°C for 30 min.

[0066] Furthermore, in step S13, the washing solution 1 contains 40-50% v / v acetonitrile, 0.2-0.3 M sodium chloride, and 5-6% v / v formic acid; the washing solution 2 contains 25-30% v / v acetonitrile and 0.1-0.2% v / v formic acid; preferably, the washing solution 1 contains 50% v / v acetonitrile, 0.2 M sodium chloride, and 6% v / v formic acid; and the washing solution 2 contains 30% v / v acetonitrile and 0.1% v / v formic acid.

[0067] Further, in step S13, after washing, the eluent is eluted at 25-37°C for 20-30 min on a shaker, and the eluent is collected. The eluent contains 8-10% v / v ammonia; preferably, the eluent is eluted for 30 min and contains 10% v / v ammonia.

[0068] Furthermore, in step S13, the eluent is lyophilized to obtain lyophilized phosphopeptides, and the lyophilized phosphopeptides are resuspended in a 1-2% v / v formic acid aqueous solution for high pH reversed-phase chromatography fractionation or desalting; preferably, a 1% v / v formic acid aqueous solution is used.

[0069] Furthermore, in step S13, the mass spectrometry detection includes data acquisition modes of data-dependent acquisition (DDA) and data-independent acquisition (DIA) and a step of quantitative data analysis. The quantitative data analysis uses statistically common significance testing methods, such as the Empirical Bayes t-test or Student's t-test, or one or more of these. The thresholds for Foldchange and p-value are not limited to a single value and need to be selected according to different situations.

[0070] Thirdly, the present invention provides a site-level analysis method, comprising the following steps:

[0071] S21: Constructing a solid support for coupled enzymatically hydrolyzed peptides, comprising: Method 1: firstly, coupling the labeled protein obtained by the neighboring protein labeling method of claim 1 with a solid support containing specific groups, and then subjecting it to reduction, alkylation reaction and enzymatic hydrolysis, and collecting the solid support for coupled enzymatically hydrolyzed peptides; or Method 2: firstly, subjecting the labeled protein obtained by the neighboring protein labeling method of claim 1 to reduction, alkylation reaction and enzymatic hydrolysis, and then coupling it with a solid support containing specific groups, and collecting the solid support for coupled enzymatically hydrolyzed peptides;

[0072] S22: React the solid support of the coupled enzyme-hydrolyzed peptide with a hydroxylamine-containing reagent, collect the release solution, desalt or fractionate by high-pH reversed-phase chromatography, and then detect by mass spectrometry.

[0073] The specific groups include hydrazide and / or hydroxylamine groups, and the solid support includes magnetic beads, resin or agarose.

[0074] Further, in step S21, method 1 is the same as step S11 of the protein level analysis method, and the solid support of the coupled enzymatically digested peptides is collected after enzymatic digestion.

[0075] Furthermore, in step S21, method 2 includes the following steps:

[0076] S211: Add dithiothreitol to the labeled protein solution and shake to reduce it, then add iodoacetamide and shake to alkylate it, thus obtaining the labeled protein after reduction and alkylation.

[0077] S212: Dilute the labeled protein from the reduction and alkylation reaction in step S211 with ammonium bicarbonate, dilute the urea to below 1 M, add trypsin for enzymatic digestion, collect the enzymatically digested peptides, desalt and freeze-dry;

[0078] S213: Resuspend the lyophilized enzymatically digested peptides in enrichment buffer 2, mix with a solid support containing specific groups, and oscillate to obtain a solid support for the coupled enzymatically digested peptides.

[0079] The enrichment buffer 2 contains citrate-sodium citrate buffer with a pH of 4.4-4.6.

[0080] Further, in step S211, when the catalyst-protein system is an extracellular catalyst-protein system, the labeled protein solution is a labeled protein resuspension of the labeled protein in urea; when the catalyst-protein system is an intracellular catalyst-protein system, the labeled protein solution is labeled cells resuspended in urea, followed by cell lysis to obtain the labeled protein solution.

[0081] Furthermore, in step S211, the concentration of the urea is 2-8 M, preferably 8 M.

[0082] Further, in step S211, the final concentration of the dithiothreitol is 10-20 mM, preferably 10 mM; and the final concentration of the iodoacetamide is 20-40 mM, preferably 20 mM.

[0083] Furthermore, in step S211, the reduction reaction temperature is 37~56℃ and the time is 1~2 h, preferably 56℃ and the time is 1 h; the alkylation reaction is incubated at room temperature in the dark for 30~40 min, preferably 30 min.

[0084] Further, in step S212, the concentration of ammonium bicarbonate is 20~100 mM, preferably 100 mM.

[0085] Further, in step S212, the mass ratio of trypsin to protein is 1:(20~50), preferably 1:25.

[0086] Furthermore, in step S212, enzymatic hydrolysis is performed at 25~37℃ for 16 hours or more, preferably at 37℃ for 16 hours.

[0087] Further, in step S213, when the catalyst is a peroxidase, the ratio of the initial protein (labeled protein) of the enzymatically hydrolyzed peptide to the solid support with specific groups is 1~3 mg: (50~100) μL. When the catalyst-protein system is a catalyst-protein extracellular system, it is preferably 1.5 mg: 100 μL; when the catalyst-protein system is a catalyst-protein intracellular system, it is preferably 3 mg: 100 μL.

[0088] Further, in step S213, when the catalyst is a photocatalyst, the ratio of the initial protein amount (labeled protein) of the enzymatically hydrolyzed peptide to the solid support with specific groups is 0.1~3 mg: (10~100) μL. When the catalyst-protein system is a catalyst-protein extracellular system, it is preferably 0.2 mg: 15 μL; when the catalyst-protein system is a catalyst-protein intracellular system, it is preferably 3 mg: 100 μL.

[0089] Furthermore, in step S213, the oscillation reaction temperature is 25~37℃ and the time is more than 20 h, preferably the oscillation reaction temperature is 37℃.

[0090] Further, in step S22, the solid support of the coupled enzyme-hydrolyzed peptides is washed with sodium chloride, acetonitrile and sodium acetate respectively to remove non-specifically adsorbed peptides, and then reacted with a reagent containing hydroxylamine with the solid support of the coupled enzyme-hydrolyzed peptides.

[0091] Furthermore, in step S22, the solid support for the coupled enzymatically hydrolyzed peptides is washed with 1-2 M sodium chloride, 50-80% v / v acetonitrile, and 40-60 mM sodium acetate, respectively; preferably 1.5 M sodium chloride, 80% v / v acetonitrile, and 50 mM sodium acetate.

[0092] Furthermore, the hydroxylamine-containing reagent contains 150-250 mM hydroxylamine compound, 40-60 mM sodium acetate, and 0.5-1% w / v aniline; the hydroxylamine compound includes methoxyamine hydrochloride and / or hydroxylamine hydrochloride; preferably, the hydroxylamine-containing reagent contains 200 mM methoxyamine hydrochloride, 50 mM sodium acetate, and 1% w / v aniline.

[0093] Furthermore, in step S22, the reaction is carried out at 25~37°C for more than 16 hours on an oscillator, preferably at 37°C.

[0094] Furthermore, in step S22, the mass spectrometry detection includes data acquisition modes of data-dependent acquisition (DDA) and data-independent acquisition (DIA) and a step of quantitative data analysis. The quantitative data analysis uses statistically common significance testing methods, such as the Empirical Bayes t-test or Student's t-test, or one or more of these. The thresholds for Foldchange and p-value are not limited to a single value and need to be selected according to different situations.

[0095] Fourthly, the present invention provides an optical protein detection method, in which the labeled protein obtained by the adjacent protein labeling method is reacted with a fluorescent dye containing hydrazide and / or hydroxylamine groups, reduced by sodium cyanoborohydride, and subjected to fluorescence imaging analysis.

[0096] Alternatively, the labeled protein obtained by the adjacent protein labeling method is reacted with biotin containing hydrazide and / or hydroxylamine groups, reduced by sodium cyanoborohydride, and then bound to a streptavidin-labeled HRP antibody for immunoblotting analysis.

[0097] Furthermore, the fluorescence imaging analysis method includes the following steps:

[0098] S31: Fix the labeled cells with Carnoy's fixative and permeabilize them with PBST solution (PBS buffer containing Triton X-100 or Tween-20);

[0099] S32: Place the cells treated in step S31 in a citrate-sodium citrate buffer, add a fluorescent dye containing an acylhydrazine group or a hydroxylamine group and sodium cyanoborohydride, and incubate.

[0100] S33: Block the cells treated with S32 with a blocking solution containing goat serum, Tween-20 and PBS buffer.

[0101] S34: Incubate the cells treated in step S33 with the primary antibody against the target protein, and wash the cells with PBST solution;

[0102] S35: Incubate the cells treated in step S34 with the fluorescent secondary antibody and 4',6-diamidindo-2-phenylindole, wash the cells with PBST solution, and perform fluorescence imaging analysis.

[0103] Furthermore, in step S31, the fixed time is 15~20 min, preferably 15 min.

[0104] Furthermore, in step S31, the concentration of Triton X-100 in the PBST solution is 0.5~0.6% v / v, and the permeation treatment time is 30~60 min; preferably, the concentration of Triton X-100 in the PBST solution is 0.5% v / v, and the permeation treatment time is 30 min.

[0105] Furthermore, in step S32, the pH of the citrate-sodium citrate buffer solution is pH 4.4~4.6.

[0106] Furthermore, in step S32, the final concentration of the fluorescent dye containing hydrazide or hydroxylamine groups is 0.05~0.2 mM, and the final concentration of sodium cyanoborohydride is 0.3~1.2 mM; preferably, the final concentration of the hydrazide / hydroxylamine fluorescent probe is 0.1 mM, and the final concentration of sodium cyanoborohydride is 0.6 mM.

[0107] Furthermore, in step S32, the incubation is carried out at 4°C to 37°C in the dark for 16 to 20 hours, preferably at 4°C in the dark for 20 hours.

[0108] Furthermore, in step S33, the room is sealed in the dark for 1-2 hours, preferably 1 hour.

[0109] Furthermore, in step S33, the blocking solution contains 3-5% v / v goat serum, 0.08-0.12% v / v Tween-20 and PBS buffer; preferably, the blocking solution contains 5% v / v goat serum, 0.1% v / v Tween-20 and PBS buffer.

[0110] Furthermore, in step S34, the incubation is carried out at 4°C in the dark for 10-12 hours, preferably 12 hours.

[0111] Furthermore, in steps S34 and S35, the cells are washed three times, with each washing session lasting 10 to 15 minutes, preferably 15 minutes.

[0112] Furthermore, in steps S34 and S35, the concentration of Tween-20 in the PBST solution is 0.08~0.12% v / v, preferably 0.1% v / v.

[0113] Furthermore, in the immunoblotting analysis method, the final concentration of biotin containing hydrazide or hydroxylamine groups is 0.5-2 mM, and the final concentration of sodium cyanoborohydride is 3-12 mM; preferably, the final concentration of biotin containing hydrazide or hydroxylamine groups is 1 mM, and the final concentration of sodium cyanoborohydride is 6 mM.

[0114] Furthermore, in the immunoblotting analysis method, the labeled protein is incubated with biotin containing hydrazide or hydroxylamine groups and sodium cyanoborohydride at a temperature of 25-37°C for 16-20 h; preferably at 37°C for 20 h.

[0115] Compared with the prior art, the present invention has the following beneficial effects:

[0116] (1) Higher site identification coverage and sensitivity

[0117] Due to the strong affinity between biotin probes and streptavidin, the recovery rate of labeled peptides using the traditional proximity labeling method (BP-APEX) is unsatisfactory, making it difficult to identify labeled proteomes using site-level analysis (Site ID). Although subsequent improved APEX site-specific methods have offered some improvement in site identification, the results remain unsatisfactory. The Ald-PL / Ket-PL technology of this invention utilizes highly reactive aldehyde / keto-phenol probes combined with a chemically reversible acylhydrazine / hydroxylamine enrichment process to achieve highly sensitive and comprehensive labeled site identification. Using these labeled sites, the proteome can be directly and clearly labeled, overcoming the false positive rate problem introduced by protein-level analysis (Protein ID) and also solving the problem of insufficient coverage in site-level analysis (Site ID) of previous methods. With its efficient site-level analysis, Ald-PL / Ket-PL can provide more comprehensive identification coverage in mapping subcellular membrane protein topologies and higher sensitivity in protein dynamic conformation analysis, enabling not only more accurate identification of labeled proteomes but also more sensitive protein structure resolution.

[0118] (2) Less disturbance to cells

[0119] The aldehyde / keto-phenol probe used in this invention exhibits stronger reactivity. In the peroxidase-protein system, it maintains highly efficient peroxidase labeling reactions even at extremely low H2O2 concentrations (50-100 μM), and can also perform live-cell peroxidase-catalyzed labeling under low oxidative perturbation (100 μM H2O2). Compared with traditional peroxidase labeling methods, this method reduces the amount of H2O2 required by 10 times. In the photocatalyst-protein system, the aldehyde / keto-phenol probe can achieve efficient labeling under visible light irradiation (blue light, 420-430 nm), avoiding the introduction of H2O2. This greatly reduces or avoids H2O2-induced cytotoxicity and perturbation of endogenous post-translational modifications (PTMs), overcoming the limitations of traditional proximity labeling methods such as site-level analysis and cellular physiological perturbation. It can accurately map subcellular PTM dynamics in redox-sensitive biological systems and under near-physiological conditions, further expanding the application scope of proximity protein labeling methods.

[0120] (3) Simple operation and good reproducibility

[0121] The aldehyde / keto-phenol probes used in this invention are commercially available and do not require chemical synthesis. The solid-phase support for reversible enrichment of acylhydrazides / hydroxylamines is also commercially available, and the standardized reversible enrichment procedure for acylhydrazides / hydroxylamines ensures the simplification and reproducibility of the experiment.

[0122] (4) High compatibility of labeled peptide mass spectrometry

[0123] This invention utilizes a chemically reversible hydrazide / hydroxylamine enrichment technique to covalently capture proteins or peptides modified with aldehyde / keto-phenol probes. After undergoing stringent washing conditions (to remove non-specific adsorption and reduce background interference during detection), the labeled peptides are recovered using hydroxylamine. Compared to the larger molecular structure of biotin tags, the peptides recovered after hydroxylamine recovery have smaller tag molecules, reducing the generation of more fragment ions, which is beneficial for spectral analysis and further improves site identification efficiency.

[0124] (5) The data results are highly reliable and accurate.

[0125] Compared to previous methods, the method of this invention significantly improves both site identification efficiency and coverage. Site-level analysis (Site ID) allows direct identification of labeled proteomes using labeled sites, resulting in more accurate and reliable identification results without the need for a control group. It overcomes the high false-positive rate caused by non-specific protein binding in traditional proximity labeling methods such as protein ID. Attached Figure Description

[0126] Figure 1 The relevant technical principles and advantages of the technical solution of this invention are explained.

[0127] Figure 2 This study describes protein labeling in a peroxidase-protein extracellular system under non-denaturing conditions using proximity protein labeling (Ald-PL) based on aldehyde-phenol probes (salicylaldehyde, 3-hydroxybenzaldehyde, and p-hydroxybenzaldehyde). In the diagram, A is a flowchart illustrating the peroxidase (APEX2) catalytic labeling of aldehyde-phenol probes (using p-hydroxybenzaldehyde as an example) in cell lysate. Free proteins in cell lysates were labeled with the p-hydroxybenzaldehyde probe APEX2. The labeled proteins were digested with trypsin, and the digested peptides underwent reversible enrichment by acylhydrazine and release of methoxyhydroxylamine before being analyzed by mass spectrometry. B is a bar chart (n=3) showing the number of labeled peptides and proteins identified by the three aldehyde-phenol probes (salicylaldehyde, 3-hydroxybenzaldehyde, and p-hydroxybenzaldehyde) at the cell lysate level. C is a Venn diagram showing the labeled peptides identified by the three aldehyde-phenol probes (salicylaldehyde, 3-hydroxybenzaldehyde, and p-hydroxybenzaldehyde) at the cell lysate level. D is a Venn diagram showing the labeled proteins identified by the three aldehyde-phenol probes (salicylaldehyde, 3-hydroxybenzaldehyde, and p-hydroxybenzaldehyde) at the cell lysate level through the labeled peptides.

[0128] Figure 3This study investigates protein labeling in a photocatalyst-protein extracellular system using Ald-PL (Alg-Phenolic Probe-Based Labeling) technology under non-denaturing conditions. In the diagram, A shows the chemical structures of riboflavin and eosin Y. B shows the number of peptides labeled by the aldehyde-phenol probe (taking p-hydroxybenzaldehyde as an example) in different photocatalysts (riboflavin and eosin Y) under blue light (420–430 nm) irradiation.

[0129] Figure 4 This diagram illustrates the use of Ald-PL (Algol-phenol Probe-Based Proteometry) for mitochondrial proteomics analysis. A shows the workflow of site-level mitochondrial proteomics analysis using Ald-PL. B shows a HEK293T cell system stably expressing matrix-APEX2 (molecularly localized APEX2) labeled with p-hydroxybenzaldehyde and analyzed using confocal fluorescence imaging. 5-FAM hydrazide (5-FAM hydrazide HCl, a fluorescent dye) was used to visualize the p-hydroxybenzaldehyde-labeled protein (green, 488 nm); anti-TOMM20 (a mitochondrial marker protein) was used to visualize mitochondrial localization (red, 561 nm); scale bar, 10 μm. C shows a comparison of the labeling sites of p-hydroxybenzaldehyde (p-Hba), biotin-phenol (BP), and dethiobiotin-phenol (DBP) probes using Ald-PL.

[0130] Figure 5 This diagram illustrates the use of Ald-PL (Algol-phenol Proximity Protein Labeling) technology to identify the topological structure of mitochondrial membrane proteins. A shows a volcano diagram (Matrix vs. OMM) of the mitochondrial membrane protein topological structure resolved by Ald-PL; B shows the site matching accuracy analysis of Ald-PL technology for mitochondrial transmembrane (TM) proteins (IMM-TM and OMM-TM) with known topological structures; C is a schematic diagram of topological structure verification (known topological domains) of the inner mitochondrial membrane transmembrane protein (IMM-TM) using Ald-PL technology.

[0131] Figure 6This study uses Ald-PL (Alg-Phenol Probe-Based Labeling) to reveal the dynamic conformational changes of the mitochondrial proteome under CCCP stimulation. A is a volcano plot showing the dynamic conformational changes of mitochondrial proteins under 20 μM CCCP stimulation using Ald-PL; B is a volcano plot showing the dynamic conformational changes of mitochondrial proteins under 50 μM CCCP stimulation using Ald-PL; C is a Venn diagram showing significantly upregulated labeling sites under CCCP stimulation (20 and 50 μM); D is a Venn diagram showing significantly downregulated labeling sites under CCCP stimulation (20 and 50 μM); and E is a heatmap showing significantly changed (fold change > 1.41, p-value < 0.05) labeling sites identified under both 20 and 50 μM CCCP stimulation. Red indicates upregulation of the labeling site in the CCCP stimulation group (log2 fold change) relative to the control group, and blue indicates downregulation. Detailed Implementation

[0132] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0133] like Figure 1 The diagram illustrates the relevant technical principles and advantages of the present invention.

[0134] Ald-PL / Ket-PL: Full name: Aldehyde / Ketone-phenol Probe-based Proximity Labeling; Chinese definition: Proximity labeling technology based on aldehyde / ketone-phenol probes; Description: A peroxidase or photocatalytic labeling technology that combines highly reactive aldehyde / ketone-phenol probes with a chemically reversible acylhydrazine / hydroxylamine enrichment process. It can be used to identify subcellular proteomes, explore protein-protein interaction dynamics, map subcellular membrane protein topologies, and analyze protein dynamic conformational changes under low concentrations of hydrogen peroxide or without hydrogen peroxide through various analytical strategies (mass spectrometry, fluorescence imaging, and immunoblotting).

[0135] BP-APEX: Full name: Biotin-phenol (BP)-based APEX proximity labeling; Chinese definition: APEX proximity labeling technology based on biotin-phenol; Description: An APEX proximity labeling technology that utilizes biotin-phenol (BP) probes and streptavidin enrichment, which can be used for subcellular proteomics and protein-protein interaction analysis through protein level analysis (Protein ID).

[0136] Click-APEX: Full name: Clickable (Alk-Ph)-APEX proximity labeling; Chinese definition: APEX proximity labeling technology based on click chemistry; Description: A site-specific APEX proximity labeling technology that uses an alkynyl-phenol (Alk-ph) probe to introduce biotin through a click chemical reaction and bind it to streptavidin enrichment and UV cleavage. It can be used to map subcellular membrane protein topologies through site-level analysis (Site ID).

[0137] DBP-APEX: Full name: Desthiobiotin-phenol (DBP)-based APEX proximity labeling, DBP-APEX; Chinese definition: APEX proximity labeling technology based on desthiobiotin-phenol (DBP); Description: A site-specific APEX proximity labeling technology that utilizes desthiobiotin-phenol (DBP) probes and streptavidin enrichment, which can be used to map subcellular membrane protein topologies through site-level analysis (Site ID).

[0138] Protein ID: Full name: Protein-level identification and quantitation analysis; Chinese definition: Protein-level analysis; Description: Protein-level analysis (Protein ID) is a high-throughput proteomics technology based on mass spectrometry (MS) detection. It uses the peptides obtained from protein hydrolysis and digestion for mass spectrometry analysis to identify the protein composition in complex biological samples.

[0139] Site ID: Full name: Site-level identification and quantification analysis; Chinese definition: Site-level analysis; Description: Site-level analysis (Site ID) is a high-resolution proteomics technique based on mass spectrometry (MS) used to accurately identify modification sites on proteins (post-translational modifications or small molecule probe-labeled sites) and quantify their abundance changes.

[0140] APEX-EM: Full name: Engineered Ascorbate Peroxidase-Mediated Electron Microscopy Staining; Chinese definition: Engineered ascorbate peroxidase (APEX) mediated electron microscopy staining technique; Description: This is a nanoscale electron microscopy imaging technique based on APEX enzyme-catalyzed DAB in situ polymerization, which can accurately resolve the topological structure of membrane proteins.

[0141] APEX-Pattern: Full name: APEX-based Spatial Biotinylation Pattern Analysis; Chinese explanation: Spatial biotinylation pattern analysis based on APEX; Description: This is an imaging technique that accurately characterizes the topological structure of membrane proteins based on the different biotinylation labeling patterns (diffused / restricted) generated by APEX enzymes in living cells.

[0142] DIA: Full name: Data-Independent Acquisition; Chinese meaning: Data-independent acquisition; Description: A mass spectrometry acquisition mode that can systematically detect all peptides in a sample, improving data coverage and quantitative accuracy.

[0143] DDA: Full name: Data-Dependent Acquisition; Chinese definition: Data-dependent acquisition; Description: A mass spectrometry acquisition mode that analyzes the peptide signal intensity in the primary mass spectrometer in real time, selects the ion with the highest intensity for secondary fragmentation and detection, and is suitable for efficient identification of target peptides.

[0144] Example 1: In vitro protein labeling under non-denaturing conditions in a peroxidase-protein system using aldehyde / keto-phenol probe-based neighboring protein labeling technology (Ald-PL / Ket-PL).

[0145] In this embodiment, salicylaldehyde, 3-hydroxybenzaldehyde, and p-hydroxybenzaldehyde were used as representative aldehyde / keto-phenol probes, and purified APEX2 was used as an exogenously added peroxidase. Combined with the reversible enrichment of acylhydrazine and the release of methoxyhydroxylamine, as well as multiple analytical strategies (mass spectrometry and immunoblotting), the protein composition and enzymatic labeling site information of the labeled protein can be obtained, providing an effective analytical means for the study of protein conformational changes and protein-protein interactions.

[0146] I. Obtaining the labeled protein

[0147] 1. Resuspend the cultured cells (HeLa cells) in PBS. Cell lysis can be performed by liquid nitrogen freeze-thaw or sonication. Collect the cell lysis products and dissolve them in PBS (10 mM) to obtain a protein solution. No denaturing agents are added during this process.

[0148] 2. Mix the protein solution from step 1 with exogenously added purified peroxidase (APEX2) to make the final peroxidase concentration 0.5 mg / ml.

[0149] 3. In step 2, add the aldehyde-phenol probe (final concentration 4 mM, completely dissolved in PBS), incubate for 2 min, then add H2O2 (final concentration 100 μM) to initiate enzymatic labeling, and react at 37℃ for 3 min to obtain the labeled group. The sample without the aldehyde-phenol probe is used as the control group.

[0150] 4. Immediately after the labeling treatment in step 3, add the termination reagent (final working concentration of 10 mM sodium azide, 10 mM sodium ascorbate and 5 mM quinoline dimethacrylate) to the reaction system and place it on ice for 10 min to terminate the reaction; add cold acetone at a ratio of 1:5 (v / v) (terminated solution: acetone, 1:5) and precipitate the labeled protein overnight at -30℃.

[0151] II. Reversible enrichment of hydrazides

[0152] 1. Reversible enrichment of protein-layer acylhydrazides:

[0153] (1) Resuspend the labeled protein from step 4 in enrichment buffer 1 (citric acid-sodium citrate buffer with 4% w / v sodium dodecyl sulfate SDS, pH 4.4-4.6), and sonicate the solution until clear and transparent using an ultrasonic homogenizer. Test the protein concentration of the protein solution using the BCA method. Take 1.5 mg of protein (3-4 μg / μL) from each sample for subsequent enrichment. Perform at least 3 technical replicates for each sample.

[0154] (2) Mix the protein solution with 100 μL of hydrazide resin and react at 37°C for more than 20 h on a shaker.

[0155] (3) The hydrazide resin treated in step (2) was collected in 8 M urea, and dithiothreitol (DTT, final concentration 10 mM) was added and reacted at 56 °C for 1 h on a shaker. Then iodoacetamide (IAA, final concentration 20 mM) was added and incubated at room temperature in the dark for 30 min on a shaker.

[0156] (4) Washing steps: Wash the acylhydrazine resin treated in step (3) four times with 8 M urea, 1.5 M sodium chloride and 100 mM ammonium bicarbonate respectively to remove non-specifically adsorbed proteins.

[0157] (5) On-bead hydrolysis: Add trypsin (trypsin: protein = 1:25, mass ratio) to the acylhydrazine resin treated in step (4) in 100 mM ammonium bicarbonate (600 μL) and hydrolyze for more than 16 h at 37°C on a shaker. Separate the acylhydrazine resin by centrifugation (1000g×5 min) and collect the on-bead hydrolysate.

[0158] (6) Collect the on-bead enzymatic hydrolysate treated in step (5), add 1% (v / v) trifluoroacetic acid (TFA) for acidification and desalting, and use it for mass spectrometry detection to identify the composition of the labeled protein. The desalting step is as follows: take out the packing material and sieve plate from the commercial 60 mg HLB column (Waters), trim the original sieve plate to a diameter of 1.5 mm, and fill it and the packing material (5 mg per sample) into a 200 μL pipette tip to make a miniature "solid phase extraction column" for desalting. After activation with acetonitrile once, equilibration with 0.1% (v / v) trifluoroacetic acid aqueous solution twice, loading the sample (this step is acidified on-bead enzymatic hydrolysate), rinsing twice with 0.1% (v / v) formic acid (FA) aqueous solution, and eluting twice with 80% (v / v) ACN and 0.1% (v / v) FA, and collect the eluent. All solutions were loaded onto the column in 200 μL volumes each time. The liquid could be centrifuged or dripped down using a syringe. The eluent was then freeze-dried in a freeze dryer, reconstituted, and analyzed by mass spectrometry.

[0159] (7) Collect the hydrazide resin treated in step (5) and wash it four times with 1.5 M sodium chloride, 80% (v / v) acetonitrile and 50 mM sodium acetate to remove non-specifically adsorbed peptides.

[0160] (8) Add methoxyhydroxylamine release solution (composed of 200 mM methoxylamine hydrochloride, 50 mM sodium acetate and 1% w / v aniline) to the hydrazide resin treated in step (7) and react at 37°C for more than 16 h on a shaker.

[0161] (9) Collect the release liquid from step (8), desalt it according to the method in step (6) (no acidification required), freeze dry it, reconstitute it and perform mass spectrometry detection to identify the labeling site information.

[0162] 2. Reversible enrichment of peptides by layered acylhydrazides:

[0163] (1) Replace the enrichment buffer 1 in the protein layer hydrazide reversible enrichment step (1) with 8 M urea, and the other steps are the same.

[0164] (2) Add DTT (final concentration 10 mM) to the protein solution in step (1) and incubate at 56°C for 1 h. Then add IAA (final concentration 20 mM) and incubate at room temperature in the dark for 30 min.

[0165] (3) Dilute the protein solution treated in step (2) with 100 mM ammonium bicarbonate (NH4HCO3), dilute the 8 M urea in it to below 1 M, and then add trypsin (trypsin:protein = 1:25, mass ratio) and enzymatically hydrolyze at 37℃ for more than 16 h. The enzymatically hydrolyzed peptides are acidified, desalted and lyophilized to obtain enzymatically hydrolyzed peptides. The desalting step is the same as step (6) of the protein layer hydrazide reversible enrichment. Desalting is performed using a 60 mg commercial HLB column (Waters) (no need to make a micro "solid phase extraction column"). The volume of all solutions loaded onto the column is 1 mL each time. The liquid flows down naturally by gravity. Collect the eluent and lyophilize it in a freeze dryer.

[0166] (4) Resuspend the enzymatically digested peptide (1.5 mg of initial protein) from step (3) in enrichment buffer 2 (citric acid-sodium citrate buffer, pH 4.4~4.6), mix with 100 μL of hydrazide resin, and react at 37°C for more than 20 h on a shaker.

[0167] (5) Washing step: Collect the acyl hydrazine resin treated in step (4), and after treatment according to steps (7) to (9) of protein-level reversible enrichment of acyl hydrazine, it is used for mass spectrometry detection.

[0168] III. Mass Spectrometry Detection and Data Processing:

[0169] 1. Protein ID Analysis:

[0170] (1) The acidified and desalted on-bead enzyme hydrolysate was detected and analyzed by DDA mode mass spectrometry.

[0171] (2) The raw files obtained by mass spectrometry detection and analysis were searched in the pFind software for proteomics analysis, and the corresponding quantitative methods were set to obtain the identification results of the labeled proteins, thus realizing the protein composition analysis of the labeled proteins.

[0172] 2. Site ID Analysis:

[0173] (1) DDA mode mass spectrometry detection and analysis of the desalted release solution.

[0174] (2) The raw files obtained from mass spectrometry analysis were searched using the pFind software for proteomics analysis. The corresponding quantitative methods were set to obtain the quantitative results of labeled peptides and sites. Protein hierarchical enrichment can also be used for site-level analysis (Site ID), but site-level analysis results are usually obtained using peptide hierarchical enrichment methods.

[0175] IV. Immunoblot Analysis:

[0176] In the protein solution of the protein layer reversible enrichment step (1) of protein layer acylhydrazide, acylhydrazide biotin (final concentration 1 mM) and sodium cyanoborohydride (final concentration 6 mM) were added and incubated at 37°C for 20 h. Immunoblot analysis was performed, and the labeled protein was detected by streptavidin-labeled HRP antibody.

[0177] V. Experimental Results

[0178] This invention selects three aldehyde / keto-phenol probes (salicylaldehyde, 3-hydroxybenzaldehyde, and p-hydroxybenzaldehyde) for in vitro enzymatic labeling. The specific procedure is as follows: Figure 2 As shown in Figure A, after cell lysis, three aldehyde / keto-phenol probes were used to label free proteins under peroxidase-catalyzed conditions at a low concentration of H2O2 (100 μM). After protein digestion, the digested peptides underwent a reversible enrichment process using peptide hierarchical acylhydrazide and a methoxyhydroxylamine release procedure. The released peptides were then used for mass spectrometry analysis. The results showed that ( Figure 2 The p-hydroxybenzaldehyde probe (B in the original text) showed significantly better labeling performance in cell lysates than other structural analogs, identifying 1633 modified peptides covering 843 labeled proteins. Comparative analysis with other probes revealed (…). Figure 2 C and Figure 2 The p-hydroxybenzaldehyde probe (D in the original text) exhibits significant advantages in peptide and protein labeling coverage, likely due to the para-substitution effect. Despite their similar structures, para-substitution may be more conducive to covalent labeling with exposed tyrosine residues, reducing steric hindrance and enhancing enzymatic labeling accessibility. Compared to other peroxidase labeling methods, this approach achieves higher site identification coverage and sensitivity, and can characterize protein conformational changes and protein-protein interactions through differences in enzymatic labeling accessibility, providing an effective analytical tool for structural biology, drug target discovery, and protein-protein interaction networks.

[0179] Example 2: In vitro protein labeling under non-denaturing conditions in a photocatalyst-protein system using aldehyde / keto-phenol probe-based neighboring protein labeling technology (Ald-PL / Ket-PL).

[0180] In this embodiment, p-hydroxybenzaldehyde is used as a representative aldehyde / keto-phenol probe, and eosin Y or riboflavin is used as an exogenously added photocatalyst. Combined with the reversible enrichment of acylhydrazine and the release of methoxyhydroxylamine, as well as a variety of analytical strategies (mass spectrometry and immunoblotting), the protein composition and labeling site information of the labeled protein can be obtained, providing an effective analytical means for the study of protein conformational changes and protein-protein interactions.

[0181] I. Obtaining the labeled protein

[0182] 1. Resuspend the cultured cells (K562 cells) in PBS. Cell lysis can be performed by liquid nitrogen freeze-thaw or sonication. Collect the cell lysis products and dissolve them in PBS (10 mM) to obtain a protein solution. No denaturing agents are added during this process.

[0183] 2. Mix the protein solution from step 1 with the exogenously added photocatalysts (riboflavin and eosin Y) to make the final concentration of riboflavin or eosin Y 10 μM.

[0184] 3. Add the aldehyde-phenol probe (final concentration 4 mM, completely dissolved in PBS) to step 2, incubate for 2 min, start photocatalytic labeling under blue light (420~430 nm) irradiation, and react at 25℃ for 10 min.

[0185] 4. After the labeling treatment in step 3, stop the blue light irradiation to terminate the reaction; add cold acetone at a ratio of 1:5 (v / v) (the solution after termination: acetone, 1:5), and precipitate the labeled protein overnight at -30℃.

[0186] II. Reversible enrichment of hydrazides

[0187] 1. Reversible enrichment of protein-layer acylhydrazides:

[0188] (1) Resuspend the labeled protein from step 4 in enrichment buffer 1 (citric acid-sodium citrate buffer with 4% w / v sodium dodecyl sulfate SDS, pH 4.4-4.6), and sonicate the solution until clear and transparent using an ultrasonic homogenizer. Test the protein concentration of the protein solution using the BCA method. Take 0.2 mg of protein (0.4-1 μg / μL) from each sample for subsequent enrichment. Perform at least 3 technical replicates for each sample.

[0189] (2) Mix the protein solution with 15 μL of hydrazide resin and react at 37°C for more than 20 h on a shaker.

[0190] (3) The subsequent steps are the protein layer hydrazide reversible enrichment (3) to (9) in step two of Example 1, and the enzyme hydrolysate and release solution are collected.

[0191] 2. Reversible enrichment of peptides by layered acylhydrazides:

[0192] The process is the same as the reversible enrichment of peptides by layered acylhydrazine (1) to (5) in step two of Example 1. In step (3), the enzymatically hydrolyzed peptides are desalted using the micro "solid-phase extraction column" of Example 1. In step (4), the starting protein amount of the enzymatically hydrolyzed peptides is 0.2 mg, the volume of acylhydrazine resin is 15 μL, and the reaction is carried out at 37°C for more than 20 h on a shaker.

[0193] III. Mass Spectrometry Detection and Data Processing:

[0194] Mass spectrometry detection and data processing for in vitro protein conformational changes and protein-protein interaction analysis were the same as in Example 1. Protein ID analysis was used to identify the protein composition of the labeled protein. Site ID analysis was used to identify the quantification results of the labeled peptides and sites.

[0195] IV. Immunoblot Analysis:

[0196] The specific steps are the same as step four of Example 1.

[0197] V. Experimental Results

[0198] This invention selects p-hydroxybenzaldehyde as a representative aldehyde / ketone-phenol probe for in vitro photocatalytic labeling. After cell lysis, p-hydroxybenzaldehyde, under blue light (420~430 nm) irradiation, reacts with riboflavin or eosin Y (… Figure 3 In step A), the protein is catalyzed into an active free radical to form a covalent label with the free protein. After protein digestion, the digested peptides undergo a reversible enrichment process using peptide-layer acylhydrazine and a methoxyhydroxylamine release process. The released peptides are then used for mass spectrometry analysis. The results show that ( Figure 3 In this study, 1792 and 1060 labeled peptides of p-hydroxybenzaldehyde were identified using riboflavin and eosin Y as photocatalysts, respectively, achieving good identification results. Compared with traditional proximity labeling methods, this method avoids the introduction of H2O2, has less disturbance to cell physiology, and can obtain higher site identification coverage and sensitivity. It can characterize protein conformational changes and protein-protein interactions by the difference in accessibility of labeled sites.

[0199] Example 3: Analysis of subcellular proteome and protein-protein interactions using aldehyde / keto-phenol probe-based neighboring protein labeling technology (Ald-PL / Ket-PL).

[0200] In this embodiment, p-hydroxybenzaldehyde is used as a representative aldehyde / keto-phenol probe to target the engineered APEX2 peroxidase to the target subcellular region. Combined with the reversible enrichment of acylhydrazine and the release of methoxyhydroxylamine, as well as a variety of analytical strategies (mass spectrometry, fluorescence imaging and immunoblotting), a new technical platform is provided for the dynamic monitoring of subcellular proteome and protein-protein interaction changes.

[0201] I. Obtaining labeled cells

[0202] 1. HEK293T cells stably expressing Matrix-APEX2 (APEX2 targets the mitochondrial matrix, mito-V5-APEX2) or NES-APEX2 (APEX2 targets the cytoplasm) were cultured in 10 cm × 10 cm culture dishes.

[0203] 2. Add p-hydroxybenzaldehyde (final concentration 4 mM, completely dissolved in the culture medium) to the cells from step 1 and incubate for 30 min. Add H2O2 (100 μM) to initiate enzymatic labeling and react at 37℃ for 3 min. This serves as the mitochondrial labeling group. HEK293T cells stably expressing NES-APEX2 serve as the cytoplasmic labeling control group.

[0204] 3. After the labeling treatment in step 2, immediately discard the reaction solution, add the stop solution (PBS buffer containing 10 mM sodium azide, 10 mM sodium ascorbate and 5 mM quinoline dimethacrylate), place on ice for 10 min to stop the reaction.

[0205] 4. After terminating the reaction in step 3, discard the stop solution, add PBS buffer, suspend the cells in the culture dish by pipetting, and collect them in a 2 mL centrifuge tube. Discard the supernatant, add PBS buffer again to wash the cells. Repeat this washing step 3 times to ensure that excess probes are washed away.

[0206] II. Enriched Proteins:

[0207] 1. Protein ID Analysis:

[0208] (1) Resuspend the cells in enrichment buffer 1 and lyse the cells using an ultrasonic homogenizer to obtain a protein solution. The protein concentration of the protein solution is tested by the BCA method. Take 3 mg of protein from each sample for enrichment. Perform at least 3 technical replicates for each sample group.

[0209] (2) Process the protein layer hydrazide reversible enrichment solution according to the method of (2) to (6) in Example 1, collect the on-bead enzyme digestion solution, and use it for subsequent protein post-translational modification enrichment or acidification desalting for mass spectrometry detection.

[0210] 2. Site ID Analysis:

[0211] (1) Cells were resuspended in 8 M urea and lysed using an ultrasonic homogenizer to obtain a protein solution. The protein concentration of the protein solution was tested using the BCA method. 3 mg of protein from each sample was used for subsequent enzymatic digestion. Each sample group was tested in at least 3 replicates.

[0212] (2) Process according to the steps (2) to (5) of the peptide layering acylhydrazine reversible enrichment in Example 1, collect the release liquid, and desalt the release liquid (step (6) of the protein layering acylhydrazine reversible enrichment in Example 1, the release liquid does not need to be acidified) for mass spectrometry detection (DDA and DIA analysis). Alternatively, it can be divided into 6 fractions by high pH reverse phase chromatography (no acidification required) for mass spectrometry detection (DDA analysis).

[0213] The high-pH reversed-phase chromatography fractionation procedure is as follows: First, prepare mobile phase A (acetonitrile) and mobile phase B (10 mM NH4HCO3). Take 10 mg of HLB packing material and sonicate it in a 50% water bath for 5 min. Then, pack the packing material and sieve plate (1.5 mm diameter) into a 200 μL pipette tip to create a miniature "solid-phase extraction column". Wash once with a mixture of 80% (v / v) mobile phase A + 20% (v / v) mobile phase B and acetonitrile, and then wash twice with 0.1% (v / v) formic acid aqueous solution. Load the sample (release solution) into the miniature "solid-phase extraction column", and wash twice with 0.1% (v / v) formic acid aqueous solution. Elute once with a gradient of mobile phases of different concentrations. The ratios of each mobile phase in the gradient elution are shown in Table 1. Collect the eluent from each elution. The eluents from gradient elutions 1 and 7 were mixed, as were those from gradient elutions 2 and 8, 3 and 9, 4 and 10, and 5 and 11. The eluent from gradient elution 6 was collected, divided into six fractions, lyophilized, and then analyzed by MS. The loading volume for all solutions was 200 μL each time.

[0214] Table 1

[0215]

[0216] 3. Post-translational modification group analysis (e.g., phosphorylation):

[0217] (1) Collect the on-bead enzyme hydrolysate, mix the on-bead enzyme hydrolysate (500 μL) with the enrichment solution (composed of 80% (v / v) acetonitrile and 6% (v / v) formic acid) at a volume ratio of 1:1, and then add 10 mg Ti 4+ -IMAC microspheres were reacted at 25°C for 2 h on an oscillator to enrich phosphopeptides.

[0218] (2) Washing steps: Collect the phosphopeptides treated in step (1) and wash them for 30 min at 25°C on a shaker with washing solution 1 (composed of 50% (v / v) acetonitrile, 0.2 M sodium chloride and 6% (v / v) formic acid) and washing solution 2 (composed of 30% (v / v) acetonitrile and 0.1% (v / v) formic acid).

[0219] (3) After washing in step (2), add eluent (10% (v / v) ammonia) and elute at 25°C for 30 min on a shaker. Collect the eluent and freeze-dry to obtain freeze-dried phosphopeptides. Resuspend the freeze-dried phosphopeptides in 1% (v / v) formic acid aqueous solution and fractionate them into 6 fractions by high pH reversed-phase chromatography for mass spectrometry detection (DDA analysis).

[0220] III. Mass Spectrometry Detection and Data Processing:

[0221] 1. Protein ID Analysis:

[0222] (1) The acidified and desalted on-bead enzyme hydrolysate was detected and analyzed by DIA mode mass spectrometry.

[0223] (2) The raw files obtained from mass spectrometry analysis were searched using the Spectronaut proteomics analysis software. Corresponding quantitative methods were set to obtain protein quantification results. Significance tests were performed, and proteins with significant changes were identified based on Fold change and p-value. Compared to the control group (cytoplasmic marker group), the mitochondrial marker group showed more significant enrichment of mitochondrial proteome.

[0224] 2. Site ID Analysis:

[0225] (1) The six fractions after fractionation in step 2 were analyzed by DDA mode mass spectrometry.

[0226] (2) The raw files obtained from mass spectrometry analysis were searched using the pFind proteomics analysis software. Corresponding quantitative methods were set to obtain quantitative results for labeled peptides and sites. No background information subtraction was required from the control group, and the identified labeled peptides exhibited high subcellular specificity.

[0227] (3) Use DIA mode mass spectrometry to detect and analyze the sample after desalting the release solution in step 2.

[0228] (4) The raw files obtained from mass spectrometry analysis were searched using the proteomics analysis software Spectronaut. The mitochondrial marker site database (established in Example 4) was loaded, and the corresponding quantification method was set to obtain the quantitative results of the labeled peptides and sites. No background signal subtraction was required from the control group, and the identified labeled peptides exhibited high subcellular specificity.

[0229] 3. Post-translational modification group analysis (e.g., phosphorylation):

[0230] (1) The six fractions after fractionation in step 3 were analyzed by DDA mode mass spectrometry.

[0231] (2) The raw files obtained from mass spectrometry analysis were searched using the pFind software for proteomics analysis. The corresponding quantitative methods were set to obtain the quantitative results of subcellular phosphorylated peptides and sites.

[0232] IV. Fluorescence Imaging Analysis:

[0233] 1. After terminating the reaction of cells (cultured in 2 cm × 2 cm glass-bottomed dishes) in step one, fix them with Carnoy's fixative at room temperature for 15 min, and then permeabilize them in 0.5% (v / v) PBST solution (PBS buffer containing 0.5% v / v Triton X-100) at room temperature for 30 min.

[0234] 2. Place the cells in enrichment buffer 2, add 5-FAM hydrazide (5-FAM hydrazide HCl, hydrazide fluorescent dye, dissolved in DMSO, final concentration 0.1 mM) and sodium cyanoborohydride (final concentration 0.6 mM), and incubate at 4°C in the dark for 20 h.

[0235] 3. After incubation, block in blocking solution (5% v / v goat serum diluted in PBS buffer containing 0.1% v / v Tween-20) at room temperature in the dark for 1 h.

[0236] 4. After blocking treatment, the cells were incubated with the primary antibody of the target protein (mitochondrial marker, anti-TOMM20, mitochondrial outer membrane translocase 20) at 4°C in the dark for 12 h. The cells were then washed three times with 0.1% (v / v) PBST solution (PBS buffer containing 0.1% v / v Tween-20) for 15 min each time.

[0237] 5. Cells were incubated with fluorescent secondary antibody (Alexa Fluor 488 / 594 labeled secondary antibody) and DAPI (nuclear dye) at room temperature in the dark for 1 h. Cells were then washed three times with 0.1% (v / v) PBST for 15 min each time. Fluorescence imaging analysis was then performed to visualize the colocalization of the aldehyde-phenol probe-labeled protein and the target protein.

[0238] V. Immunoblot Analysis:

[0239] Cells treated in step one were resuspended in enrichment buffer 1 and lysed using an ultrasonic homogenizer to obtain a protein solution. The protein concentration of the protein solution was determined by the BCA method. Acid hydrazide biotin (final concentration 1 mM) and sodium cyanoborohydride (6 mM) were added to the protein solution and incubated at 37°C for 20 h. The labeled proteins were then analyzed by Western blotting using a streptavidin-labeled HRP antibody. Each group had the same protein content.

[0240] VI. Experimental Results

[0241] like Figure 4As shown in Figure A, HEK293T cells stably expressing APEX2 in the mitochondrial matrix (Matrix-APEX2) were used as model cells, and a workflow for mitochondrial proteomics analysis using Ald-PL technology was designed. Fluorescence imaging analysis ( Figure 4 (B) shows that the p-hydroxybenzaldehyde-labeled protein exhibits a strong co-localization trend with mitochondria, demonstrating the high labeling selectivity of Ald-PL technology in live cell labeling. Compared with previous BP and DBP probes ( Figure 4 The number of labeled sites identified by Ald-PL was significantly improved (C in the original text). Under DIA-MS analysis, Ald-PL identified 714 labeled sites (with a specificity of 93.9%), demonstrating significantly better site coverage than biotin probes (BP, 268; DBP, 379). Compared to traditional protein ID-dependent proximity labeling methods, Ald-PL's site ID analysis showed higher identification accuracy and eliminated the need for a control group. These results demonstrate that Ald-PL technology can achieve highly sensitive and comprehensive labeling site identification under low oxidative perturbation conditions, representing a major breakthrough in the field of proximity labeling. It also overcomes the limitations of traditional methods in terms of cellular physiological perturbation and site-level analysis, providing a powerful tool for in-depth research into the dynamic regulatory networks of organelles.

[0242] Example 4: Identification of subcellular membrane protein topology using aldehyde / keto-phenol probe-based neighboring protein labeling technology (Ald-PL / Ket-PL).

[0243] In this embodiment, p-hydroxybenzaldehyde was used as a representative aldehyde / keto-phenol probe to target APEX2 peroxidase to different subcellular compartments (intracellular and extracellular). Combined with the reversible enrichment of acylhydrazine and the release of methoxyhydroxylamine, as well as the mass spectrometry analysis strategy, an effective tool was provided for accurately mapping the topological structure of subcellular membrane proteins.

[0244] 1. HEK293T cells stably expressing Matrix-APEX2 (mito-V5-APEX2) and HEK293T cells stably expressing OMM-APEX2 (APEX2-targeting mitochondrial outer membrane protein MAVS) were cultured separately in 10 cm × 10 cm culture dishes. Cells expressing Matrix-APEX2 were used as the mitochondrial matrix marker group, primarily marking sites in the matrix region. Cells expressing OMM-APEX2 were used as the mitochondrial outer membrane marker group, primarily marking sites in and outside the mitochondrial intermembrane lumen. Each group of cells was treated according to steps 2-4 of step one in Example 3 to obtain labeled cells.

[0245] 2. Following the Site ID analysis method in step two of Example 3, cells were processed, and the release solution was collected. The release solution of each sample was divided into two equal portions. One portion was desalted (no acidification required) and lyophilized. The lyophilized peptides were reconstituted with 0.1% (v / v) formic acid (FA) solution for quantitative mass spectrometry analysis. The other portion was obtained by mixing the release solutions of samples from repeated techniques (mixing only with the corresponding labeling groups) to obtain a mixture of Matrix-APEX2 and OMM-APEX2 labeling groups. This mixture was then fractionated using the high-pH reversed-phase chromatography method of Example 3 (no acidification required) to establish a mitochondrial labeling site database. The Matrix-APEX2 labeling group consisted of 6 fractions, and the OMM-APEX2 labeling group consisted of 6 fractions, for a total of 12 fractions.

[0246] 3. Mass spectrometry detection and data processing for mitochondrial membrane protein topological structure analysis:

[0247] (1) Quantitative mass spectrometry analysis of mitochondrial matrix and outer membrane markers was performed using DIA mode.

[0248] (2) Mass spectrometry was used to detect the mitochondrial matrix and outer membrane markers in high pH reverse phase chromatography fractions (12 fractions) to establish a mitochondrial marker site database.

[0249] (3) The raw files obtained from mass spectrometry analysis were searched using the proteomics analysis software Spectronaut. The established mitochondrial marker site database was loaded, and the corresponding quantitative methods were set to obtain the quantitative results of the marker sites. The significance test was performed, and the marker sites with significant changes were determined based on the Fold change and p-value. These differentially expressed marker sites were matched with the corresponding membrane protein topology to determine the membrane protein topology.

[0250] 4. Experimental Results

[0251] In Example 4, engineered APEX2 was targeted to different compartments (matrix or outer membrane) of the mitochondria. However, this is not the only example; the target organelle can be the mitochondria, endoplasmic reticulum, Golgi apparatus, cell membrane, or other organelles. Using Ald-PL technology, two different mitochondrial APEX2-targeted cell lines (Matrix-APEX2 and OMM-APEX2 HEK293T cells) were labeled, and quantitative mass spectrometry analysis was used to screen for significantly different marker sites. Figure 5(A) These sites exhibit different labeling trends. For example, Matrix-APEX primarily labels the mitochondrial matrix region, while OMM-AEPX mainly labels the mitochondrial intermembranous lumen, endoplasmic reticulum, and cytoplasm. Comparison and validation of these significantly different sites with proteins containing known membrane topological domains showed a site matching accuracy exceeding 90%, demonstrating the reliability of Ald-PL technology for large-scale identification of membrane protein topology. Figure 5 (B in the text). Compared with other membrane topology identification methods (APEX-EM, APEX-Pattern, Click-APEX, and DBP APEX), Ald-PL technology has higher identification coverage and sensitivity, and can be used for large-scale exploration of unknown membrane protein topologies, providing a powerful tool for a deeper understanding of their molecular functions and mechanisms of action. Figure 5 C in the diagram represents the site matching results of identifying the topological structure (known topological domain) of transmembrane proteins in the inner mitochondrial membrane using Ald-PL technology. This provides an important basis for experimental verification and can also provide a reference model for predicting the structure of membrane proteins in unknown topological domains, thus assisting in drawing more accurate mitochondrial membrane protein maps.

[0252] Example 5: Analysis of Subcellular Proteome Dynamic Conformation Changes Using Ald-PL / Ket-PL Probe-Based Proximity Labeling Technology

[0253] In this embodiment, p-hydroxybenzaldehyde was used as a representative aldehyde / keto-phenol probe to target the engineered APEX2 peroxidase to the target subcellular region. Combining the reversible enrichment of acylhydrazine and the release of methoxyhydroxylamine with mass spectrometry analysis, the conformational changes of the subcellular proteome under different physiological states were monitored by utilizing differences in the accessibility of enzymatic labels. HEK293T, which stably expresses APEX2 (Matrix-APEX2) in the mitochondrial matrix, was used as the research model.

[0254] 1. HEK293T cells stably expressing Matrix-APEX2 were subjected to either no CCCP (Carbonylcyanide 3-chlorophenylhydrazone) stimulation or CCCP stimulation (20 and 50 μM) for 4 h at 37℃. The control group was the one without CCCP stimulation, and the stimulation group was the one after CCCP stimulation.

[0255] 2. Following the Site ID analysis method in step two of Example 3, process the cells and collect and process the release fluid.

[0256] 3. Mass spectrometry detection and data processing for subcellular proteome dynamic conformational change analysis are the same as in Example 4. Quantitative mass spectrometry analysis is performed to obtain significantly changed marker sites. These significantly changed marker sites are used to characterize the microscopic changes and local conformational alterations of proteins. Subsequent verification of conformational changes of proteins of interest can be achieved by combining other methods (restriction protease digestion, APEX-EM).

[0257] 4. Experimental Results

[0258] In Example 5, the stimulating drug used was CCCP, including but not limited to drug stimulation or other types of stimulation conditions. Different concentrations (e.g., low, medium, and high doses) of the test drug (such as CCCP and FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) and other mitochondrial stress inducers, or other target drugs) can be selected to treat cells according to research needs. Stimulation conditions such as oxidative stress, starvation, and temperature changes can also be selected for the stimulation group to study protein conformational changes under different physiological / pathological conditions. Taking a cell line stably expressing APEX2 in the mitochondrial matrix as an example, the dynamic conformational changes of the mitochondrial proteome were studied under CCCP stimulation. The HEK293T cell line stably expressing Matrix-APEX2 was used for no stimulation or CCCP stimulation (20 and 50 μM), respectively. Using Ald-PL technology, quantitative mass spectrometry analysis was used to screen for significantly different marker sites. Upregulated marker sites were defined as sites that became more exposed after stimulation, and downregulated sites were defined as sites that became more occult after stimulation. Following CCCP stimulation, local protein conformation may change, leading to alterations in probe labeling site accessibility and resulting in different labeling trends. This allows for large-scale screening of potential conformational change candidates. Using Ald-PL technology, 309 and 284 significant labeling sites were identified after CCCP stimulation (20 μM and 50 μM), respectively, including 173 and 142 upregulated sites and 136 and 142 downregulated sites. Figure 6 A and Figure 6 (B in the text). These significant marker sites have high overlap and their variation trends are highly consistent, demonstrating the reproducibility and reliability of the data. Figure 6 C in Figure 6 D and Figure 6 (E in the text).

[0259] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method of labeling a proximity protein, the method comprising, An aldehyde / ketone-phenol small molecule probe is used as a substrate, a catalyst is used to catalyze the substrate into an active free radical-containing substrate, the active free radical-containing substrate forms a covalent label with a target protein, and a labeled protein is obtained; The catalyst is a peroxidase or a photocatalyst; The catalyst and / or the target protein are located in a catalyst-protein system; The catalyst-protein system includes a catalyst-protein extracellular system or a catalyst-protein intracellular system; The catalyst-protein extracellular system is a mixture containing the catalyst and the target protein; The catalyst-protein intracellular system is a cell containing a fusion protein of a peroxidase and the target protein. Or a cell containing a subcellular-targeted photocatalyst and the target protein.

2. The proximity protein labeling method of claim 1, wherein, The aldehyde / ketone-phenol small molecule probe is an aromatic compound with a phenolic hydroxyl group, an aldehyde group, or a ketone group as a skeleton structure, and simultaneously contains a phenolic hydroxyl group, an aldehyde group, or a ketone group functional group in the structure; preferably, the small molecule probe includes at least one of salicylaldehyde, 3-hydroxybenzaldehyde, p-hydroxybenzaldehyde, 2,6-dimethyl-4-hydroxybenzaldehyde, and p-hydroxyacetophenone.

3. The proximity protein labeling method of claim 1, wherein, When the catalyst is a peroxidase, H2O2 is added at a final concentration of 10-1000 μM to initiate peroxidase catalysis labeling.

4. The proximity protein labeling method of claim 1, wherein, The peroxidase includes horseradish peroxidase or ascorbate peroxidase.

5. The proximity protein labeling method of claim 1, wherein, The labeling time is 1-10 min, and the labeling temperature is 25-37°C.

6. The proximity protein labeling method of claim 1, wherein, When the catalyst is a photocatalyst, visible light with a wavelength of 380-780 nm is used to initiate photocatalyst catalysis labeling.

7. The proximity protein labeling method of claim 1, wherein, The photocatalyst includes an organic small molecule photocatalyst or a transition metal photocatalyst; Preferably, the organic small molecule photocatalyst includes riboflavin and / or eosin Y, and the transition metal photocatalyst includes a ruthenium catalyst.

8. A method of protein level analysis, characterized by, The method includes the following steps: S11: Constructing a solid support for coupling a labeled protein, coupling the labeled protein obtained by the adjacent protein labeling method of claim 1 with a solid support with a specific group, reducing, alkylating, and enzymatically digesting, and collecting an enzymatic digestion solution; S12: Acidifying and desalting the enzymatic digestion solution of step S11, and directly detecting by mass spectrometry; or, S13: using Ti 4+ The immobilized metal affinity chromatography polymer microspheres are used to enrich the phosphopeptides in the enzymatic hydrolysate of step S11, and the phosphopeptides are eluted, the eluate is collected, desalted or fractionated by high pH reverse phase chromatography, and mass spectrometry is performed. The specific group includes a hydrazide and / or a hydroxylamine group, and the solid support includes a magnetic bead, a resin, or agarose.

9. A site level analysis method characterized by, The method includes the following steps: S21: Constructing a solid support for coupling an enzymatic digestion peptide segment, including method 1: first coupling the labeled protein obtained by the adjacent protein labeling method of claim 1 with a solid support with a specific group, then reducing, alkylating, and enzymatically digesting, and collecting a solid support for coupling an enzymatic digestion peptide segment; or method 2: first reducing, alkylating, and enzymatically digesting the labeled protein obtained by the adjacent protein labeling method of claim 1, and then coupling with a solid support with a specific group, and collecting a solid support for coupling an enzymatic digestion peptide segment; S22: Reacting a reagent containing hydroxylamine with the solid support for coupling an enzymatic digestion peptide segment, collecting a release solution, desalting or fractionating by high-pH reverse phase chromatography, and detecting by mass spectrometry; The specific group includes a hydrazide and / or a hydroxylamine group, and the solid support includes a magnetic bead, a resin, or agarose.

10. An optical-based protein detection method, characterized by, The labeled protein obtained by the method of claim 1 is reacted with a fluorescent dye containing a hydrazine and / or hydroxyl amine group, reduced by sodium cyanoborohydride, and subjected to fluorescent imaging analysis. Or, the labeled protein obtained by the method of claim 1 is reacted with biotin containing a hydrazine and / or hydroxyl amine group, reduced by sodium cyanoborohydride, combined with streptavidin labeled HRP antibody, and subjected to immunoblotting analysis.

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