CN118206565A

CN118206565A - Catalytic marking reagent with phosphatidylserine targeting function and micro-region proteome marking and identifying method

Info

Publication number: CN118206565A
Application number: CN202211615338.9A
Authority: CN
Inventors: 张丽华; 王鹤; 江波; 蒋倩倩; 赵宝锋; 杨开广; 张玉奎
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2024-06-18

Abstract

The invention discloses a catalytic marking reagent with a phosphoserine targeting function and a micro-regional proteome marking and identifying method, wherein the catalytic marking reagent with the phosphoserine targeting function is utilized to transfer an enrichment probe connected with the reagent to nucleophilic amino acid of protein nearby the phosphoserine, so that the phosphoserine micro-regional protein is covalently marked with biotin, and the stronger the affinity between the reagent and the phosphoserine is, the higher the marking reaction efficiency is. And further, after enrichment by the streptavidin agarose spheres, performing liquid chromatography-mass spectrometry analysis to realize in-situ marking and identification of the phosphatidylserine micro-region proteome. The method is oriented to proteomics analysis of subcellular microenvironments, chemical reaction is carried out by guiding a labeled reagent to specific microenvironments and microdomains by using limited space distance, a powerful tool is provided for identifying subcellular structures such as vesicles or related proteomes of the microenvironments in cells, and the method has the advantages of small disturbance, good specificity, high reaction efficiency, good spatial resolution and the like.

Description

Catalytic marking reagent with phosphatidylserine targeting function and micro-region proteome marking and identifying method

Technical Field

The invention relates to a catalytic marking reagent with a phosphatidylserine targeting function and a phosphatidylserine related micro-area proteome marking and identifying method, belonging to the field of biochemistry.

Background

Eukaryotic cell interiors are highly modular and functional, including a variety of subcellular structures such as organelles, vesicles, and protein aggregates that rely on different protein compositions to perform specific biological functions. The localization and expression levels of proteins vary with changes in the environment, and their changes within a specific space-time mediate precise regulation of cellular signal networks, such as assembly and degradation of protein complexes, translocation of transcription factors in the cytoplasm and nucleus, and redistribution of proteins under cellular stress, etc. (Science 2009,326,1220-1224). Thus elucidating the spatiotemporal dynamics of subcellular proteomes is crucial for a better understanding of various biological processes.

Phosphatidylserine (phosphatidylserine, ptdSer) is an important component of eukaryotic cell membranes, accounting for about 10% of the total lipids of the cell, and is the most abundant anionic phospholipid in eukaryotic cells. PtdSer is particularly abundant in cell membranes compared to other biological membranes (Cell Communication AND SIGNALING 2019,17,126). PtdSer is an important signaling molecule on a series of pathways such as cell senescence, death and coagulation, and plays an important role in signaling during life. In healthy cells, ptdSer is typically distributed inside the cell membrane. When the cells undergo apoptosis due to exogenous and exogenous stimuli, ptdSer turns from the inner side to the outer side of the cell membrane, thereby recruiting macrophages as 'eatme' signal molecules and promoting the clearance of apoptotic cells (Nature Reviews Immunology 2014,14,166-180). In the micro-region enriched in PtdSer on the membrane, a large number of proteins with special physicochemical properties are distributed, and the PtdSer related dynamic turnover and signal transduction are involved.

The distribution of phosphatidylserine on the membrane is balanced by the synergistic effect of the invertase (flippase) and the promiscuous enzyme (scramblases). The invertase transfers PtdSer from the outer surface to the inner surface of the plasma membrane, holding it in the inner surface. The promiscuous enzyme nonspecifically and bidirectionally transports PtdSer between the inner surface and the outer surface of the plasma membrane, so that the distribution of the promiscuous enzyme tends to be symmetrical. In normal cells, the invertase (ATP 11C) continuously inverts phosphatidylserine (PtdSer) on the plasma membrane, whereas the promiscuous enzymes (TMEM 16F and Xkr) are inactive. In apoptotic cells, caspases cleave ATP11C and Xkr, which inactivate invertase and promiscuous enzyme respectively, resulting in irreversible PtdSer exposure (TRENDS IN CELL Biology 2015,25,639-650). In the tumor microenvironment, ptdSer is abnormally exposed on the outer surfaces of tumor cells, vascular endothelial cells and microvesicles, which helps to form an overall immunosuppressive signal, antagonizes the development of local and systemic anti-tumor immune processes, and assists tumor cells in immune escape (ImmunoTargets AND THERAPY 2018,7,1-14). However, little is known about the specific mechanism by which PtdSer in tumor cells is externalized to the cell surface, and the key proteins involved in this process.

Inhibition of cell surface exposure of PtdSer in tumor microenvironments may be a novel approach to enhance tumor immune responses. The protein of PtdSer micro-area on the surface of tumor cells is marked by adopting chemical active molecules in situ, and PtdSer dynamic change related proteome is identified and analyzed in living cells, so that the method has important significance for explaining PtdSer mediated immunosuppression and molecular mechanism thereof, and can provide new insight for tumor immunotherapy.

Disclosure of Invention

The application provides a catalytic marking reagent with a phosphatidylserine targeting function, which consists of a phosphatidylserine targeting group, namely a bis-zinc dimethyl pyridine amine and a catalytic reaction group, namely pyridine oxime or N-acyl-N-alkyl sulfonamide. The pyridine oxime catalytic reagent needs to work in conjunction with an N-acyl-N-acyl donor. The reagent is focused on a specific cell membrane region by targeting phosphatidylserine, so that an enrichment probe connected to the reagent is transferred to a nucleophilic amino acid of a protein near the phosphatidylserine through an SN2 reaction, and the phosphatidylserine micro-region protein is covalently labeled with biotin. Enrichment is carried out by streptavidin agarose microsphere, mass spectrum data acquisition is carried out by a liquid chromatography-mass spectrometry system, and identification and analysis are carried out on the phosphoserine micro-region proteome.

The technical scheme adopted by the method is as follows:

A catalytic labelling reagent with a phosphatidylserine targeting function, wherein the catalytic labelling reagent comprises a compound containing a biszinc dimethyl pyridine amine group; the catalytic marking reagent also comprises an N-acyl-N-alkyl sulfonamide active structure.

Optionally, the compound containing the biszinc dimethyl pyridine amine group is formed by connecting the biszinc dimethyl pyridine amine group with a pyridine oxime group; the catalytic labelling reagent further comprises an N-acyl-N-alkyl sulfonamide group as a donor.

Optionally, the preparation method of the compound containing the biszinc dimethyl pyridine amine group comprises the following steps:

2,2' -methylpyridine amine was reacted with N- (t-butoxycarbonyl) -L-tyrosine methyl ester to give Dpa-Boc.

Removing Boc groups in the Dpa-Boc to obtain Dpa;

After 3-bromopropionic acid reacts with pyridine-4-formaldehyde oxime, dpa is added for continuous reaction to obtain a product Dpa-PyOx;

And reacting the Dpa-PyOx with zinc ions to obtain the compound containing the biszinc dimethyl pyridine amine group.

Optionally, the preparation method of the N-acyl-N-alkyl sulfonamide group donor comprises the steps of reacting 4-nitrobenzene sulfonamide with D-biotin to generate a compound 2;

Reacting the compound 2 with p-nitrobenzyl bromide to obtain the N-acyl-N-alkyl sulfonamide donor.

Optionally, the compound containing the biszinc dimethyl pyridine amine group is formed by connecting the biszinc dimethyl pyridine amine group with an N-acyl-N-alkyl sulfonamide group.

reacting 2,2' -picoline amine with N- (tert-butoxycarbonyl) -L-tyrosine methyl ester to obtain Dpa-Boc;

removing Boc groups in the Dpa-Boc to obtain Dpa;

reacting Dpa with glutaric anhydride to obtain Dpa-COOH;

Reacting 4-p-aminosulfonyl benzoic acid with 1, 8-octanediamine-N-Boc to produce compound 3;

reacting the compound 3 with D-biotin to obtain a compound 4;

Removing Boc group from the compound 4, and then reacting with the Dpa-COOH to generate a compound 5;

And (3) reacting the compound 5 with zinc ions to obtain the compound containing the biszinc dimethyl pyridine amine group.

The application also provides a catalytic marking method with the phosphatidylserine targeting function, which comprises the step of carrying out contact reaction on the catalytic marking reagent with the phosphatidylserine targeting function and cells; the biszinc dimethyl pyridine amine group is tightly combined with the phosphate group in the phosphatidylserine; and the protein is marked with a probe through SN2 reaction between the N-acyl-N-alkyl sulfonamide structure and nucleophilic amino acid on the adjacent protein of phosphatidylserine on the cell membrane.

Alternatively, proteins are labeled with biotin by SN2 reaction of the N-acyl-N-alkyl sulfonamide structure with nucleophilic amino acids on proteins adjacent to phosphatidylserine on the cell membrane.

Alternatively, after completion of protein biotin labelling, cells are sonicated and then streptavidin agarose resin is added to selectively enrich the labelled cell surface proteins.

The application also provides application of the sample obtained by the marking method in enrichment and identification of in-situ proteome of phosphatidylserine specific micro-areas.

A specific implementation method of the application is set forth below.

Synthesis and characterization of PtdSer targeted catalytic labelling reagent

The final products ZnDpa-PyOx and acyl donor 1 and ZnDpa-NASA were obtained according to the following synthetic route (fig. 1). The purified product obtained in each step was subjected to chromatographic, mass spectrometric, nuclear magnetic analysis and the stability and affinity with PtdSer of the final product ZnDpa-PyOx, znDpa-NASA was characterized.

2. In vitro experiment-verification of the modification effect of marking reagents

The modification effect of the photoactivating agent was verified at the protein level. Mixing standard protein with a labeling reagent in the presence of PtdSer, and carrying out mass spectrum characterization on the labeling efficiency and the labeling site, wherein the labeling condition of the protein in the absence of PtdSer or in the presence of PBS is set.

3. Proteomics perturbation experiment

Setting a blank group and an experimental group (labeled reagent treatment), extracting proteins from cells after treatment, and obtaining peptide fragments after denaturation, reduction, alkylation and enzymolysis. And then analyzing the sample by using an ultra-high performance liquid chromatography-high resolution mass spectrometry (LC-MS/MS), screening the differential protein by using a calibration-free technology, and analyzing the disturbance condition of the catalytic marking reagent on the cell proteome.

4. Living cell labelling experiments

The reagents ZnDpa-PyOx and acyl donor 1 or ZnDpa-NASA were incubated with living cells and washed away after a certain period of time. Alexa Fluor ^TM 488 streptavidin conjugate and Cellmask ^TM dark red cell membrane dye were added and washed away after incubation for a period of time, and confocal microscopy was used to observe co-localization of the red and green markers.

The reagents ZnDpa-PyOx and acyl donor 1 or ZnDpa-NASA were incubated with living cells and washed away after a certain period of time. And (3) extracting proteins from the cells, and carrying out liquid chromatography-mass spectrometry analysis after denaturation, reduction, alkylation and enzymolysis to identify PtdSer related proteomes. The spectra were pooled using Maxquant and the proteins searched were GO annotated in uniprot and the plasma membrane proteins and cell surface proteins were analyzed for their proportion in identifying total proteins. And removing nonspecific adsorption and endogenous biotinylated proteins by using a quantitative mass spectrometry technology, screening the proteins enriched by the catalytic markers, and analyzing PtdSer related functional proteins.

The method is applied to analysis of PtdSer related dynamic protein groups in the tumor cell apoptosis process. Etoposide is used for inducing tumor cells to undergo apoptosis, normal tumor cells, early apoptosis tumor cells and late apoptosis tumor cells are respectively selected as analysis objects, and PtdSer related dynamic proteome in the tumor cell apoptosis process is identified by combining quantitative mass spectrometry technology after catalytic reagent labeling enrichment.

Binding of the targeting group to the target draws the catalytic group closer to the amino acid to be labeled, with about a strong affinity, and with higher effective concentrations of catalytic reagent for adjacent proteins, the faster the reaction efficiency.

The new method can realize the identification of in-situ proteome in living cells, focus the eyes on specific micro-areas in subcells, and realize the unprecedented spatial resolution on the level of proteome. The protein can be marked only in the presence of phosphatidylserine and a marking reagent, and the reaction has good biological orthogonality. Most of nucleophilic amino acids marked by the catalytic marking system are histidine. The mass spectrometer in the liquid chromatography-mass spectrometry system comprises an electrostatic field orbit trap (Orbitrap), a time-of-flight Tube (TOF), a triple quaternary rod (QQQ) or a Fourier transform ion cyclotron resonance mass analyzer (FT-ICR). This strategy was used for identification and analysis of the phosphoserine micro-region proteome of different biological processes in cell and tissue samples. The method can be extended to other subcellular microenvironments rich in metal ions, different pH, etc. by changing the phosphoserine targeting groups in the catalytic tag molecule.

This patent has following advantage:

(1) PtdSer targets strongly, the marking range is small, only PtdSer micro-region proteins are marked; (2) The labeling efficiency depends on the affinity of the reagent and the target, and the stronger the affinity, the higher the labeling efficiency, so that off-target effect caused by non-specific labeling is effectively avoided; (3) In ZnDpa-PyOx and acyl donor 1 labeling systems, the PyOx catalyst part can play a role repeatedly, and catalyze probes connected to the acyl donor to transfer to proteins nearby PtdSer, so that the biological orthogonality is good, but the reaction kinetics is slightly slow; in ZnDpa-NASA labelling system, one labelling reagent can only play a single role, but its reaction kinetics are fast and the binding of ZnDpa to PtdSer is in dynamic equilibrium. The combination of the two can identify more PtdSer micro-region related protein groups; (4) The method can be combined with a cross-linked mass spectrometry technology or a protein post-translational modification enrichment technology to identify the interaction protein or post-translational modification of PtdSer micro-region protein, and provides a powerful tool for finding membrane protein key enzymes or substrates involved in important life processes.

Drawings

FIG. 1 shows the structural formula (ZnDpa-PyOx, acyl donor 1, znDpa-NASA) (A) of a catalytic labelling reagent and the synthetic route (B) of the catalytic labelling reagent.

FIG. 2 is a schematic diagram of the principle and flow of labeling a micro-regional protein by a catalytic labeling system (Pr: probe, i.e. biotin).

Fig. 3 is an immunoblot experiment: the labeling effect (A) of the catalytic labeling system on living cells and the labeling effect (B) on each cell component.

Fig. 4 is a liquid chromatography-mass spectrometry analysis: the type, number and proportion of the proteins identified by the catalytic marker system (the proportion of the total identified proteins in brackets) (A) and the experimental plasma membrane proteins are involved in the analysis of biological processes.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the attached drawings: the present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are provided, but the protection scope of the present invention is not limited to the following embodiments. The raw materials used in the following examples are all commercially available.

Example 1

1. Synthesis and characterization of catalytic markers

1.1 Synthesis of catalytic labelling reagents

(1) ZnDpa-PyOx synthesis

3.37G (16.9 mmol) of 2,2' -methylpyridine amine and 0.813g (27.1 mmol) of paraformaldehyde were weighed out respectively, dissolved in 48mL of an aqueous solution of isopropyl alcohol (H ₂ O/isopropyl alcohol=5:3 (v/v)), and the pH of the above solution was adjusted to 8.0 with 1N HCl; the solution was stirred at 80 for 30min, 2.0g (6.77 mmol) of N- (t-butoxycarbonyl) -L-tyrosine methyl ester (Boc-L-Tyr-OMe) was added and the mixture was refluxed at 110 for 12h; the mixture was cooled to room temperature and the isopropanol was removed by evaporation. Then cooled on ice bath, the upper solution was decanted off, the viscous oil precipitated was dissolved in 50mL of ethyl acetate, washed with saturated NaHCO ₃ and water, respectively, and dried over anhydrous Na ₂SO₄. After vacuum drying, purification was performed using a SiO ₂ packed column (mobile phase CH ₂Cl₂/methanol/triethylamine=30:1:0.1 (v/v/v)) to give Dpa-Boc as a tan oil. The resulting Dpa3.30 g (4.60 mmol) was dissolved in anhydrous CH ₂Cl₂ (20 mL) and trifluoroacetic acid TFA (20 mL), and then stirred at room temperature for 2h; the reaction was dried by spin-drying, redissolved in 20mL CH ₂Cl₂, and repeated 3 times to remove excess TFA. Dpa is obtained after drying in vacuo as a white viscous oil.

32.1Mg (0.21 mmol) of 3-bromopropionic acid, 36.8mg (0.30 mmol) of pyridine-4-carbaldehyde oxime, dissolved in 4mL of acetonitrile, and stirred at 80 under reflux for about 2.5h were weighed. Then, 47.5mg (0.25 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 28mg (0.24 mmol) of N-hydroxysuccinimide (NHS) were added thereto, and the reaction was stirred at room temperature. After 1.5h, 75mg of Dpa (0.12 mmol,1mg/mL in DMF) was added and the reaction was continued for 1h. The reaction product was purified by semi-preparative liquid phase (Hanbon Sci & Tech, NP7000 serials pump, NU3000 serials UV-video detector) (C18 reverse phase chromatography; mobile phase: phase B acetonitrile, phase A water with 0.1% TFA; A: B=95:5 (0 min) →95:5 (30 min) →70:30 (80 min), product ZnDpa-PyOx peaked at 63 min). The product effluent fractions were collected and lyophilized to give a colorless transparent solid.

(2) Synthesis of acyl donor 1

Each of 507.4mg (2.51 mmol) of 4-nitrobenzenesulfonamide, 722.3mg (2.96 mmol) of D-biotin, 949.1mg (4.95 mmol) of EDC. HCl, 608.2mg (4.98 mmol) of 4-Dimethylaminopyridine (DMAP), and the mixture was dissolved in 12mL of DMF, and 1.25mL of DIEA was added thereto and the mixture was stirred at room temperature overnight. Semi-preparative separation of the reaction mixture (mobile phase, a: b=70:30 (0 min) →40:60 (30 min), compound 2 peaked at 17 min), product 2 was collected and spun dry as a white solid. 80.2mg (0.18 mmol) of compound 2, 243.7mg (1.1 mmol) of p-nitrobenzyl bromide was weighed out, dissolved in 3mL of DMF, and 98. Mu.L of DIEA was added thereto and stirred at room temperature for 5 hours. Semi-preparative separation of the reaction mixture (mobile phase, A: B=50:50 (0 min) →20:80 (30 min), acyl donor 1 peaked at 17 min), collection of the product fraction acyl donor 1, and spin-drying to give a white solid.

(3) ZnDpa Synthesis of NASA

2.86G (4.64 mmol) of Dpa synthesized in the above method was weighed out and dissolved in anhydrous CH ₂Cl₂ (110 mL), and 0.635g (5.57 mmol) of glutaric anhydride was added. Stirred at 50 f, refluxed and reacted overnight. After evaporation of the solvent, dpa-COOH was obtained as a pale yellow viscous oil.

412.9Mg (2.05 mmol) of 4-p-aminosulfonyl benzoic acid, 1, 8-octanediamine-N-Boc 501.2mg (2.06 mmol), 590.1mg (3.08 mmol) of EDC. HCl, 472.1mg (3.08 mmol) of 1-hydroxybenzotriazole monohydrate, dissolved in 8mL of DMF, and DIEA1.07mL were added and stirred overnight at room temperature. The reaction mixture was dissolved in 100mL of ethyl acetate, washed with saturated sodium bicarbonate solution (×2), 5% citric acid (×1), and brine (×1), respectively, and the organic layer was left, dried over anhydrous magnesium sulfate, filtered, and dried by spin to give compound 3 as a white solid.

Compound 3 282mg (0.66 mmol), D-biotin 295.5mg (1.21 mmol), EDC. HCl 385.6mg (2.01 mmol), DMAP 41.2mg (0.34 mmol) were weighed out in 8mL DMF, DIEA 345. Mu.L was added and stirred overnight at room temperature. Semi-preparative separation of the reaction mixture (mobile phase, a: b=55:45 (0 min) →25:75 (30 min), compound 4 peaked at 17 min), collection of product fractions, and lyophilization to give a white solid.

Compound 4,78 mg (0.12 mmol) was weighed out and dissolved in 1.5mL TFA and 3mL CH ₂Cl₂ and reacted for 1h with stirring at room temperature. The reaction solvent was removed by rotary evaporation, and after adding an appropriate amount of CH ₂Cl₂, it was dried by rotary evaporation, and repeated 3 times. The resulting product was dissolved in 6mL of DMF, and Dpa-COOH 104mg (0.14 mmol), EDC. HCl 37.8mg (0.20 mmol), HOBt 29.2mg (0.19 mmol), and DIEA 200. Mu.L were added and stirred at room temperature overnight. Semi-preparative separation of the reaction mixture (mobile phase, a: b=90:10 (0 min) →50:50 (40 min), compound 5 peaked at 28 min), collection of the product fractions, lyophilization to give colorless oily liquid.

50Mg (39. Mu. Mol) of the compound was weighed into 2mL of DMF, 30. Mu.L (0.41 mmol) of iodoacetonitrile was added thereto, 35. Mu.L (0.20 mmol) of DIEA was added thereto, and the mixture was stirred at room temperature overnight. Semi-preparative separation of the reaction mixture (mobile phase, a: b=90:10 (0 min) →50:50 (40 min), compound 5 peaked at 30 min), collection of the product fractions, lyophilization to give colorless oily liquid. 25mg (19. Mu. Mol) of the product was weighed into 1mL of methanol, 11.3mg ZnNO ₃·6H₂ O (dissolved in 1mL of methanol) was slowly added dropwise, and the mixture was stirred at room temperature for reaction for 1h, and lyophilized at 4℃to give a pale yellow brown solid.

1.2 Characterization of catalytic labelling reagents

(1) Mass spectrum, nuclear magnetic characterization

The purified product from each step was dissolved in methanol or acetonitrile and molecular weight was verified using orbitrap LTQ (Thermo, USA). The purified product obtained in each step (¹ H spectrum: 5-10mg, ¹³ C spectrum: 20mg or more) was dissolved in CD ₃ OD or DMSO-d ₆, and analyzed by nuclear magnetic resonance spectroscopy (AVANCE III MHz, bruker), and the structure of the compound was further confirmed by resolution.

(2) Characterization of stability of catalytic labelling reagents

The hydrolysis efficiency of acyl donor 1 (200. Mu.M) was analyzed by liquid chromatography in 50mM HEPES buffer solution, pH 7.2, physiological conditions of 37℃and a control group was set: znDpa-PyOx (20. Mu.M) in the presence of the intermediate; the hydrolysis efficiency of ZnDpa-NASA (200. Mu.M) was analyzed by liquid chromatography under the same conditions.

(3) Characterization of the affinity of the catalytic labelling Agents to PtdSer

PtdSer (1-2 mM) was dispersed in 50mM HEPES buffer (pH 7.2), and gradually (10. Mu.L. Times.24) was added dropwise to a solution in which ZnDpa-PyOx or ZnDpa-NASA (25-100. Mu.M) was dissolved. All measurements were performed at 298K. The measured heat flow is recorded and as a function of time, and the reaction peak is converted into an enthalpy change (Δh) by integration. Binding parameters (kappa, Δh, Δs, n) were evaluated by using a software Origin (MicroCal inc.) and applying a site model. Measurement in 10mM PBS buffer (pH 7.2) was set as a control group.

Bovine Serum Albumin (BSA) was used as a model to verify the effect of the labeling reagent on protein modification.

1) Annexin V (annexin V) was dissolved in 50mM HEPES buffer (pH 7.2) at 0.1mg/mL, ptdSer (10. Mu.M) was added, and incubated for 10min. Then 20. Mu. MZnDpa-PyOx and 50. Mu.M acyl donor 1, or 20. Mu. M ZnDpa-NASA, are added and incubated at 37℃for 30min. And control groups were set without PtdSer addition and without ZnDpa-PyOx or ZnDpa-NASA addition.

2) Western Blotting: 12.5% of separation gel and concentrated gel were prepared. To the labeled annexin V, 1/5 volume of 6X SDS PAGE loading buffer was added and boiled for 5min at 95. Separation by SDS-PAGE, followed by membrane transfer, blocking with 3% BSA, incubation with streptavidin-horseradish peroxidase conjugate (Sav-HRP), and final imaging by exposure to Chemisoc ^TM XRS+ (BIO-RAD) and analysis of the intensity of the biotin bands of each sample by Image Lab ^TM software.

3) Liquid chromatography-mass spectrometry analysis: dithiothreitol (DTT) was added to the labeled annexin V to a final concentration of 50mM and boiled for 5min at 95. The protein solution (about 100. Mu.g) was transferred to FASP membranes (10 k, pre-washed once with water), and centrifuged at 16000g for 30-40min at 20. The 50mM Ammonium Bicarbonate (ABC) solution was washed 1 time. 200. Mu.L of 20mM Iodoacetamide (IAA) was added thereto, and the reaction was allowed to proceed at room temperature for 30 minutes in the dark. 16000g,20 min by centrifugation. 50mM ABC washed 3 times. 100. Mu.L of 10mM ABC solution are added, and trypsin (mass spectrometry grade, promega) is added at 1:50 (enzyme: protein, m/m), 37, and the reaction is allowed to proceed for 12-16h. The peptide solutions were lyophilized and dissolved in 0.1% aqueous fa and the resulting peptide was analyzed using Easynano HPLC and Q-ExactiveTM combined four-bar Orbitrap mass spectrometer system to identify the modification site.

3. Proteomics perturbation experiment

1) SY5Y cells were cultured in a 10cm dish with DMEM medium (containing 10% fetal calf serum and 1% penicillin streptomycin) in a 37-5% CO ₂ incubator for 24 hours or more and at a density of about 90%.

2) SY5Y cells were discarded and washed once with HBS buffer (20mM HEPES,107mM NaCl,6mM KCl,2mM CaCl ₂,1.2mM MgSO₄, 11.5mM Glucose,pH 7.4). ZnDpa-PyOx and acyl donor 1 were diluted to 10. Mu.M and 20. Mu.M with HBS buffer, respectively, or ZnDpa-NASA was diluted to 10. Mu.M with HBS buffer, added to dishes and incubated with cells for 1h in a 37, 5% CO ₂ incubator. After the end, the cells were washed 3 times with PBS. The blank groups were each replaced with 0.1% dmso for the labeling reagent.

3) And (5) collecting cells. 1ml of precooled PBS was added to scrape the cells, placed in an ice-bath centrifuge tube, and washed once with PBS. The PBS solutions were pooled, 500g,4, centrifuged for 5min, washed 2 times with PBS, and counted.

4) And (5) ultrasonic crushing. Cells were dispersed in an appropriate volume of lysate (1% SDS/PBS,1% cocktail) (about 1E7 cells corresponding to 400. Mu.L lysate), 80W, sonicated with an ultrasonic cell disruptor (Scientz-IID) for 3-5min (5 s on,10s off) until the solution became clear. The BCA method determines protein concentration.

5) And (5) denaturation and reduction. DTT was added to the protein solution to a final concentration of 50mm and boiled for 5min at 95.

6) Alkylation and enzymolysis. 100-200 μg of protein is applied to FASP membranes (10 k, pre-washed once with water), 16000g, and centrifuged at 20 for 30-40min.8M urea was washed 1 time. 200. Mu.L of 20mM Iodoacetamide (IAA) was added thereto, and the reaction was allowed to proceed at room temperature for 30 minutes in the dark. 16000g,20 min by centrifugation. 8M urea was washed 3 times. The 50mM Ammonium Bicarbonate (ABC) solution was washed 3 times. 100. Mu.L of 10mM ABC solution are added, trypsin (mass spectrometry grade, promega) is added at 1:25 or 1:50 (enzyme: protein, m/m), 37, and the reaction is allowed to proceed for 12-16h.

7) 16000G,4 centrifugation for 30-40min to give peptide solution, and washing the membrane with 50 μl of 10mM ABC solution for 2 times, combining, lyophilizing, and storing at-80.

8) NanoLC-MS/MS analysis. Samples were analyzed using a four-bar Orbitrap mass spectrometer equipped with Easynano HPLC and Q-ExactiveTM in combination to construct a 1D-nano-RPLC-ESI-MS/MS system. The peptide was redissolved in 0.1% FA solution and loaded at 1-2 μg, 3 needles were repeated in parallel for each sample. Liquid chromatography conditions: mobile phase a phase: aqueous solution (volume concentration) containing 2% ACN and 0.1% fa, mobile phase B phase: an aqueous solution (volume concentration) containing 98% ACN and 0.1% fa.

9) And (5) data analysis. All data acquired by mass spectrum are searched by adopting MaxQuant-2.0.3.0 with built-in Andromeda search engine, and a calibration-free mode is set. And carrying out data processing by using Perseus _1.5.8.5 software to find out the differential protein with no calibration quantity and intensity ratio of more than 2 or 1.5.

4. Living cell labelling experiments

(1) Cell imaging test

1) SY5Y cells were cultured in 35mm glass-based confocal dishes in DMEM medium (containing 10% fetal calf serum and 1% penicillin streptomycin) for more than 24h in a 37, 5% CO ₂ incubator at a density of about 70-80%.

2) SY5Y cells were discarded and washed once with HBS buffer. ZnDpa-PyOx and acyl donor 1 were diluted to 10. Mu.M and 20. Mu.M with HBS buffer, respectively, or ZnDpa-NASA was diluted to 10. Mu.M with HBS buffer, added to dishes and incubated with cells for 1h in a 37, 5% CO ₂ incubator. The blank groups were each replaced with 0.1% dmso for the labeling reagent. Washed 3 times with PBS, added with PBS solution containing 2. Mu.g/mL Alexa Fluor ^TM 488 streptavidin conjugate, and incubated with 37, 5% CO ₂ for 1h. 10min before 1h, cellmask ^TM dark red cell membrane dye and Hoechst dye were added respectively to a final concentration of 5. Mu.g/mL and incubated for 10min at 37, 5% CO ₂. Washed 3 times with PBS, imaged under confocal microscopy (Andor, nikon Instruments inc.) and the images analyzed with Andor iQ 3.2 to compare co-localization coefficients and fluorescence intensities for each region (Alexa Fluor ^TM 488 excitation wavelength was 488nm, cellmask ^TM dark red cell membrane dye excitation wavelength was 640nm, hoechst33342 excitation wavelength was 350 nm).

(2) Immunoblotting experiment of PtdSer micro-region protein labeling effect

1) SY5Y cells were cultured in a 10cm dish with DMEM medium (containing 10% fetal calf serum and 1% penicillin streptomycin) in a 37-5% CO ₂ incubator for 24 hours or more, and when the density reached about 90%.

2) SY5Y cells were discarded and washed once with HBS buffer. ZnDpa-PyOx and acyl donor 1 were diluted to 10. Mu.M and 20. Mu.M with HBS buffer, respectively, or ZnDpa-NASA was diluted to 10. Mu.M with HBS buffer, added to dishes and incubated with cells for 1h in a 37, 5% CO ₂ incubator. Wash 3 times with PBS. Two control groups were set: ① PBS was used instead of HBS buffer; ② The labeling reagent was replaced with 0.1% dmso.

4) And (5) ultrasonic crushing. Cells were dispersed in an appropriate volume of lysate (1% SDS/PBS,1% cocktail) (about 1E7 cells corresponding to 400. Mu.L lysate), 80W, sonicated for 3-4min (5 s on,10s off) until the solution became clear. The BCA method determines protein concentration.

5) 12.5% Of separation gel and concentrated gel were prepared. 1/5 6 × SDS PAGE loading buffer was added to the protein sample and boiled for 5min at 95. Separation by SDS-PAGE, followed by membrane transfer, blocking with 3% BSA, incubation with streptavidin-horseradish peroxidase conjugate (Sav-HRP), and final imaging by exposure to Chemisoc ^TM XRS+ (BIO-RAD), image Lab ^TM software analyzed the intensity of the biotin bands for each sample (FIG. 3A), and experiments found that the biotin bands were stronger for the experimental group than for the two control groups.

6) The cell membrane and the cell nucleus, cytoplasm and other components were separated by using a Minute ^TM plasma membrane protein and a cell component separation kit (invent), and immunoblotting experiments (figure 3B) were performed respectively, and the same procedure was followed. The results show that biotin labeling occurs mainly on the plasma membrane with little apparent labeling in the cytoplasm and nucleus.

(3) Marking and identification of PtdSer micro-region proteins

2) SY5Y cells were discarded and washed once with HBS buffer. ZnDpa-PyOx and acyl donor 1 were diluted to 10. Mu.M and 20. Mu.M with HBS buffer, respectively, or ZnDpa-NASA was diluted to 10. Mu.M with HBS buffer, added to dishes and incubated with cells for 1h in a 37, 5% CO ₂ incubator. Wash 3 times with PBS. Two control groups were set: ① PBS was used instead of HBS buffer; ② The labeling reagent was replaced with 0.1% dmso. Blank (lysate) set: no reagent treatment and no enrichment.

5) Streptavidin agarose resin enrichment labeled protein. Appropriate amount of beads was removed and centrifuged at 1500g for 1min to remove the stock. 50mm ABC double column volume, three times wash, 1500g centrifugation 1min. And adding a certain amount of beads according to the protein amount, and incubating at room temperature end-to-end for 2 hours. Centrifuging at 1500g for 3min, and collecting supernatant. The beads were washed successively with 0.2% SDS/PBS (. Times.3), 1% SDS/PBS (. Times.3), PBS (. Times.3) to remove nonspecific adsorption. Eluent (2% SDS/PBS,5mM biotin) was added, incubated for 10min, eluted twice, and pooled.

6) And (3) carrying out reduction, denaturation and enzymolysis on the membrane. The eluate was transferred to FASP membranes (10 k, washed once with water beforehand) and centrifuged at 16000g for 30-40min at 20. 50mM ABC washed 3 times. Tris (2-carboxyethyl) phosphine (TCEP) was reduced at 10mM (in 50mM ABC) and reacted for 1h at 37. Leaving TCEP, IAA 20mM (dissolved in 50mM ABC) alkylated and reacted at room temperature in the dark for 30min.50mM ABC washed 3 times. Enzymatic hydrolysis was performed in 10mM ABC, trypsin was added at 1:25, 37, 12-16h. Centrifuging at 16000g 4 for 30-40min to obtain peptide solution, washing the membrane with 50 μl of 10mM ABC solution for 2 times, mixing, lyophilizing, and storing at-80.

7) NanoLC-MS/MS analysis. Samples were analyzed using a four-bar Orbitrap mass spectrometer equipped with Easynano HPLC and Q-ExactiveTM in combination to construct a 1D-nano-RPLC-ESI-MS/MS system. The peptide was redissolved in 0.1% formic acid aqueous solution and the concentration of the peptide was measured by Nanodrop and loaded at 1-2. Mu.g.

8) And (5) data analysis. All mass spectrum collected data were searched using MaxQuant _2.0.3.0 with built-in Andromeda search engine. And (3) taking proteins with unique peptides larger than or equal to 2 and scoring values larger than 30 from the library searching result, annotating on a Uniprot website, and calculating the proportion of plasma membrane proteins and cell surface proteins to total identified proteins (figure 4A), wherein the proportion of plasma membrane proteins and membrane related proteins is higher than that of a control group and lysate by an experimental group. The experimental group was also compared with the two control groups, and the non-specific marker proteins were removed by no calibration. And the screened protein is subjected to Gene Ontology analysis (figure 4B), the biological process and the signal path participated by the protein are related to PtdSer, and the result shows that the identified plasma membrane protein is involved in stress, immunity, apoptosis and other related processes and is closely related to the protein processing process.

Example 2

In the enrichment and identification of PtdSer micro-region proteomes of SY5Y cells, the cells are isotopically labeled by SILAC method to reduce errors introduced in the processing and non-standard quantification. Cell culture: SY5Y cells were cultured in a light medium containing 10% FBS and 1% penicillin-streptomycin, a light medium containing the natural amino acids L-arginine (Arg 0) and L-lysine (Lys 0), a medium containing the isotopically labeled amino acids L-arginine [ ¹³C₆ ] HCl (Arg 6) and L-lysine-4, 5-d4 (Lys 4), and a medium containing the isotopically labeled amino acid L-arginine-¹³C₆,¹⁵N₄(Arg10)and L-lysine-¹³C₆,¹⁵N₂(Lys8), respectively, under conditions of 37% CO ₂%. And (3) completely marking the cells through multiple passages to obtain SILAC light-marked, medium-marked and heavy-marked SY5Y cells. The cell treatment conditions were: light label-0.1% DMSO (in HBS), medium label-ZnDpa-PyOx (10. Mu.M) and acyl donor 1 (20. Mu.M) or ZnDpa-NASA (10. Mu.M) (in PBS), heavy label-ZnDpa-PyOx (10. Mu.M) and acyl donor 1 (20. Mu.M) or ZnDpa-NASA (10. Mu.M) (in HBS). After the cells extract proteins and measure the concentration, the experimental group and the control group are mixed with the proteins in equal amounts, and then the operations of enrichment, enzymolysis and the like are carried out, and the process is the same as that of example 1.

Example 3

The anti-cancer drug etoposide is used for inducing SY5Y cells to apoptosis, normal SY5Y cells, early-stage apoptosis SY5Y cells and late-stage apoptosis SY5Y cells are respectively selected as analysis objects, and PtdSer related dynamic proteomes in the apoptosis process of the SY5Y cells are identified by combining a quantitative mass spectrometry technology after being labeled and enriched by a catalytic reagent. The procedure is as in example 1.

Example 4

The strategy is applied to enrichment and identification of PtdSer micro-region proteome in tumor microenvironment. The obtained solid tumor tissue is digested into cells, spread in a culture dish, treated with a catalytic labeling reagent after the cells are completely adhered, and a control group is set. After marking, enrichment and enzymolysis, liquid chromatography-mass spectrometry analysis is carried out, and the process is the same as that of case 1.

Aiming at PtdSer micro-regional proteome, the invention develops a chemical proteomics method for realizing catalytic marking on living cell level, uses a catalytic marking reagent with PtdSer targeting function to perform in-situ marking and enrichment on PtdSer micro-regional protein in subcellular environment, provides a powerful tool for identifying subcellular structures such as microvesicles or related proteomes in cell internal microenvironment, and has the advantages of small disturbance, good specificity, high reaction efficiency, good spatial resolution and the like.

While the application has been described in terms of preferred embodiments, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the scope of the application, and it is intended that the application is not limited to the specific embodiments disclosed.

Claims

1. A catalytic labelling reagent with a phosphatidylserine targeting function, which is characterized in that the catalytic labelling reagent comprises a compound containing a biszinc dimethyl pyridine amine group; the catalytic marking reagent also comprises an N-acyl-N-alkyl sulfonamide active structure.

2. The catalytic labelling reagent with phosphatidylserine targeting function according to claim 1, wherein the compound containing a biszinc dimethyl pyridinamine group is formed by connecting a biszinc dimethyl pyridinamine group with a pyridinium oxime group; the catalytic labelling reagent further comprises an N-acyl-N-alkyl sulfonamide group as a donor.

3. The catalytic labelling reagent with phosphatidylserine targeting function according to claim 2, wherein the preparation method of the compound containing a biszinc dimethyl pyridine amine group comprises the following steps:

Removing Boc groups in the Dpa-Boc to obtain Dpa;

4. The catalytic labelling reagent with phosphatidylserine targeting function according to claim 2, wherein the preparation method of the N-acyl-N-alkylsulfonamide based donor comprises reacting 4-nitrobenzenesulfonamide with D-biotin to produce compound 2;

5. The catalytic labelling reagent with phosphatidylserine targeting function according to claim 1, wherein the compound containing a biszincum dimethylpyridine amine group is formed by linking a biszincum dimethylpyridine amine group with an N-acyl-N-alkyl sulfonamide group.

6. The catalytic labelling reagent with phosphatidylserine targeting function according to claim 5, wherein the preparation method of the compound containing a biszinc dimethyl pyridine amine group comprises the following steps:

removing Boc groups in the Dpa-Boc to obtain Dpa;

reacting Dpa with glutaric anhydride to obtain Dpa-COOH;

reacting the compound 3 with D-biotin to obtain a compound 4;

7. A catalytic labelling method with a phosphoserine targeting function, characterized in that a catalytic labelling reagent with a phosphoserine targeting function according to any one of claims 1 to 6 is contacted with a cell for reaction; the biszinc dimethyl pyridine amine group is tightly combined with the phosphate group in the phosphatidylserine; and the protein is marked with a probe through SN2 reaction between the N-acyl-N-alkyl sulfonamide structure and nucleophilic amino acid on the adjacent protein of phosphatidylserine on the cell membrane.

8. The method of claim 7, wherein the protein is labeled with biotin by SN2 reaction of the N-acyl-N-alkylsulfonamide structure with a nucleophilic amino acid on a protein adjacent to phosphatidylserine on the cell membrane.

9. The method of claim 8, wherein after completion of labeling the protein with biotin, the cells are sonicated and then streptavidin sepharose resin is added to selectively enrich the labeled cell surface proteins.

10. Use of a sample obtained by a labelling method according to any of claims 7 to 9 for enrichment and identification of the in situ proteome of a specific micro-region of phosphatidylserine.