CN118169263A - Cell surface proteome in-situ analysis method based on interaction of host and guest and without calibration - Google Patents

Abstract

The invention relates to a novel in-situ analysis method of cell surface proteome. In this method, a host group is modified onto a matrix support to prepare an enriched separation material. And (3) connecting the guest group with the labeling group to prepare the difunctional guest labeling probe molecule. Incubating the probe and the cells, carrying out extraction of cell whole protein by utilizing the in-situ covalent labeling of the cell surface protein of the probe, incubating the protein mixture and an enrichment material immobilized with a main molecule, and carrying out enzymolysis treatment on the enriched protein; in situ analysis of cell surface proteomes is performed in combination with liquid chromatography and quantitative proteome methods. The method has important significance for analyzing the communication among cells, the transportation of ions and other molecules and the transmission of signal cascade, understanding the paths related to diseases, immunity, drug resistance, cell function differentiation and the like, searching new disease markers and finding new action targets in drug development.

Description

In situ analysis of cell surface proteome based on host-guest interaction and label-free quantification

Technical Field

The invention relates to a novel in-situ analysis method of cell surface proteome, belonging to the technical field of biological analysis.

Background technique

Cell surface proteins refer to proteins exposed on the cell surface, including and acting as the first barrier of living cells, and are responsible for performing many key functions, including cell-to-cell interactions, signal transduction, and molecular transport. Differences in the expression of cell surface proteins are not only important markers for distinguishing different types of cells, but also the basis for phenotypic and functional differences between normal cells and diseased cells. Therefore, nearly 2/3 of drug target proteins are cell surface proteins. Identifying the cell surface proteome and studying the properties of these proteins are key steps in understanding basic biological processes and finding new targets for drug development (Expert Review of Proteomics, 2010, 7(1), 141-154; J. Sep. Sci., 2020, 43, 292–312).

Since the low abundance, high hydrophobicity and high heterogeneity limit the separation and extraction efficiency of cell surface proteins, obtaining a global spectrum of the cell surface proteome has always been a major challenge in proteomics. The rise of chemical proteomics in recent years has provided new tools for the identification of cell surface proteins, namely, chemically labeling the side chain groups of surface-exposed proteins or sugar chains (such as primary amines, carboxyl groups, and polysaccharide side chains), and then using affinity purification to separate the labeled surface proteins, such as sugar capture, metabolic labeling, and cell surface biochemical methods. The main advantage of this type of technology is that chemical labeling reagents can be customized according to the target biological structure or needs, and only a small amount of starting cells is required to obtain high-purity cell surface proteins. At present, the most commonly used method is cell surface biotinylation, that is, modifying small molecule biotin on cell surface proteins and using receptor streptavidin to specifically extract labeled proteins. However, the biotin-avidin system has the risk of contaminating the enriched protein sample. First, the interference of endogenous biotinylated proteins in cells masks low-abundance proteins and reduces the detection sensitivity; second, streptavidin itself is also a protein and will be enzymatically hydrolyzed, which may become a high-abundance contaminating protein in the sample; in addition, under high temperature or harsh washing conditions, streptavidin is also easy to denature and detach from the matrix material, resulting in the loss of enriched proteins and hindering the identification of low-abundance cell surface proteins (Nature Chemistry, 2011, 3, 154-159). Therefore, this patent uses a chemically synthesized, smaller-sized, and more physicochemically stable host-guest interaction system to analyze cell surface proteins, and obtain stable and reproducible surface proteome information with higher purity and deeper coverage.

Host-guest interaction refers to a type of supramolecular chemistry in which the host and guest molecules form inclusion complexes through non-covalent interactions. The host molecules currently under development mainly include crown ethers, cyclodextrins, cucurbiturils, pillar aromatics and calix aromatics. They specifically recognize and bind with high affinity to guest molecules such as adamantane, ferrocene, carborane and its derivatives, as well as cation- or anion-rich molecules through non-covalent interactions such as hydrophobic interactions, hydrogen bonds, electrostatic interactions, and π-π stacking, and this binding is reversible. Based on the above characteristics, host-guest interactions have been applied to many fields, such as biosensors, catalytic regulation, development of underwater adhesives, protein separation, drug delivery, etc.

In the separation of cell surface proteins, the host-guest recognition technology has a stable chemical structure, so it is not easily affected by enzymatic hydrolysis or harsh washing conditions, resulting in the loss of captured proteins, and can selectively release captured proteins by applying competitive guest molecules, which is convenient for mass spectrometry identification. In addition, due to the scalability of chemical synthesis, while being cheap and easy to obtain, the host-guest recognition molecules can also be transformed according to the different analysis purposes to improve the specificity and sensitivity of the method. Through the combination with analytical techniques such as protein immunoblotting and fluorescence imaging, it has been proved that it has higher enrichment efficiency and lower non-specific adsorption protein interference than the commonly used biotin-avidin system, and can release enriched proteins under mild conditions, and can also achieve accurate imaging of proteins (Nature Chemistry, 2011, 3, 154-159; Nature Communications, 2018, 9, 1712-1722). However, this technology has not yet been combined with mass spectrometry-based proteome quantitative analysis to analyze the cell surface proteome globally.

The label-free quantitative technology uses liquid chromatography-mass spectrometry to analyze protein enzymatic peptides. It does not require the use of expensive stable isotope labels as internal standards. It only needs to compare the signal intensity of the corresponding peptides in different samples to perform relative quantification of the proteins corresponding to the peptides. Label-free quantitative analysis can simultaneously perform quantitative analysis on multiple groups of samples, which improves the throughput of the experiment.

Therefore, in order to deeply and accurately analyze the cell surface proteome, it is possible to combine the host-guest recognition technology with high affinity and dynamic binding with mass spectrometry-based proteome label-free quantitative analysis.

In this patent, in order to address the problems of the existing biotin-avidin labeling system, such as possible contamination of enriched proteins, easy loss of low-abundance proteins, and difficulty in recovering enriched proteins, a host-guest recognition technology with the characteristics of high binding affinity, good physicochemical stability, low biological background and gentle release of labeled proteins is utilized, and it is combined with a mass spectrometry quantitative analysis method to develop an in situ analysis method of the cell surface proteome based on host-guest interaction and label-free quantification, so as to achieve in-depth and accurate analysis of cell surface proteins.

Summary of the invention

The purpose of the present invention is to provide a method for in situ analysis of cell surface proteome based on host-guest interaction and label-free quantification. The method of the present invention realizes global analysis of cell surface proteome in an in situ cell environment.

A method for in situ analysis of cell surface proteome based on host-guest interaction and label-free quantification, specifically comprising the following steps:

(1) Preparing a host-enriched material, covalently bonding the host group to a matrix material, wherein the host group is composed of one or more of crown ethers, cucurbit[n]urils (n=5-8, 10, 14), cyclodextrins, calixarene, and pillar[n]arene (n=5, 6); the matrix material is mainly one or more of agarose, polymer nanoparticles, mesoporous silica materials, carbon nanotubes, ferroferric oxide magnetic spheres, metal organic framework materials, etc.

The main enrichment material is constructed from two aspects: improving the binding affinity with the guest molecules and reducing nonspecific adsorption and potential contamination of protein samples. The surface hydrophilicity and the physicochemical stability of the material (such as resistance to acids and alkalis, detergents, high temperatures, etc.) are optimized to obtain a matrix material with low nonspecific adsorption and little sample contamination. The enrichment efficiency is improved by adjusting the bonding amount and type of the main group.

(2) preparing a guest labeled probe and connecting the guest group to the labeling group; the guest group may be one or more of ferrocene, adamantane, diamantane, polyhedral boron cluster, ammonium salt, imidazolium salt and pyridinium salt and their derivatives; the labeling group includes one or more of sulfosuccinimide ester, N-succinimide ester, acyl pyrrolidone, o-phthalaldehyde, squarate, sulfonyl fluoride, polycarbonyl, aldehyde ketone, halogenated aromatic hydrocarbon or imide ester, maleimide, 2-thiopyridine, thiosulfonate, halogenated acetyl or pyridyl disulfide, carbodiimide or isocyanate, hydrazide, phenol group, benzophenone group, phenylazide group, trifluoromethylbis(aziridine), bis(aziridine) or 2-aryl-5-carboxytetrazole, etc.

The guest-labeled probe is designed based on the principles of good membrane impermeability, biocompatibility, rapid and efficient reactivity, reaction site complementarity and high binding with the main molecule. By introducing negatively charged groups on the labeling group and using highly hydrophilic connecting arms, the probe is made difficult to penetrate the membrane and mainly reacts with cell surface proteins; labeling groups with different reaction sites and good selectivity or site-nonspecific photocross-linking labeling groups are selected, and groups that can non-covalently interact with the amino acid side chains of proteins are introduced to achieve the requirements of good biocompatibility, high reaction efficiency and site complementarity; optimization is carried out in terms of the type of guest groups and side chain groups with different charges to obtain guest-labeled probes that can be efficiently and specifically recognized by the main enrichment material.

(3) Labeling cell surface proteins with guest-labeled probes. Incubate the guest-labeled probes and cells in serum-free medium at 4°C or 37°C and 5% _CO2 for 5 minutes to 12 hours, and collect the cells. Since the probes have poor membrane permeability, they mainly accumulate on the outside of the cell membrane or between cells. The labeling group then undergoes a covalent reaction with the side chain of the nucleophilic amino acid or generates free radicals driven by ultraviolet light to insert into the XH (X＝C, N, O) bond on the protein, thereby modifying the guest group onto the cell surface protein.

(4) Disrupt the cells. Add cell lysis buffer containing 0.5-5% (v/v) protease inhibitor cocktail to the collected cells. Add 200-600 μL of lysis buffer for every 3×10 ⁶ cells. Disrupt the cells by ultrasonication. Centrifuge at 10,000-16,000 g for 5-30 minutes. Remove the supernatant to obtain a protein mixed sample.

The cell lysate comprises: 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroacetate, 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1-butyl-3-methyl-imidazolium bromide tetrafluoroborate, 1-butyl-3-methyl-imidazole chloride, 1-ethyl-3-methylimidazole chloride, 1-octyl-3-methylimidazole chloride, 1-dodecyl-3-methylimidazole chloride, 1-allyl-3-methylimidazole chloride, 1-aminopropyl-3-methylimidazole tetrafluoroborate, 1-hydroxyethyl-3-methylimidazole tetrafluoroborate, N-butylpyridine chloride, N-butylpyridine tetrafluoroborate, urea, guanidine hydrochloride, and sodium dodecyl sulfate;

The ratio of cells to cell lysis buffer was: 3 × 10 ⁶ cells added to 200-600 μl of cell lysis buffer;

The proportion of protease inhibitor cocktail in cell lysate is 0.5-5% (v/v);

The ultrasonic power is 80-160W, and the ultrasonic time is 1-5 minutes.

(5) Enrichment of cell surface proteins. The obtained protein mixed sample is mixed with the main enrichment material at a ratio of 1 mg of whole protein to 0.1-10 mg of enrichment material, and incubated at 4-37°C for 2-16 hours. The main enrichment material is fully in contact with the guest molecule on the protein and achieves high binding affinity, so that the cell surface protein modified with the guest molecule is enriched on the material; centrifuge at 1000-16000g for 5-10 minutes, discard the supernatant, and separate the cell surface protein from other intracellular proteins.

(6) Processing of the enriched proteins

The enriched protein is directly enzymatically treated on the enriched material: the protein-enriched material is dispersed in the cell lysate, dithiothreitol or tris(2-carboxyethyl)phosphine is added, the material is denatured and reduced at 25-37°C for 5 minutes to 2 hours, centrifuged at 1000-16000g for 5-10 minutes, and the supernatant is discarded; the enriched material after protein denaturation is dispersed again with the cell lysate, alkylated with iodoacetamide at 25°C for 20-40 minutes in the dark, centrifuged and the supernatant is discarded; the enriched material after protein alkylation is fully dispersed with 50mM ammonium bicarbonate, protease is added, enzymatic hydrolysis is performed at a ratio of protease to protein complex of 1:20-1:50 (w/w) for 12-16 hours, centrifuged at 1000-16000g for 5-10 minutes, and the supernatant containing the peptide sample is collected;

Alternatively, competitively elute the protein on the enriched material and pre-treat the sample with FASP. Disperse the protein-enriched material in the cell lysate, add a small guest molecule (1,1'-bis(trimethylaminomethyl)ferrocene) with stronger binding affinity to the main group, incubate at 25-95°C for 5 minutes to 2 hours, and collect the supernatant containing the protein by centrifugation; add dithiothreitol or tris(2-carboxyethyl)phosphine to the eluted protein solution, denature and reduce at high temperature, the temperature of the high temperature denaturation is 37-95°C, and the time is 5 minutes to 2 hours; transfer the sample to a 3k-10k Da filter membrane, and use iodoacetamide to alkylate in the dark for 20-40 minutes; add protease, and enzymatically hydrolyze for 12-16 hours at a ratio of protease: total protein of 1:2000-1:5000 (w/w), and collect the peptide sample by centrifugation.

The proteases used include one or more of trypsin, lysinamidase, chymotrypsin, lysine arginine N-terminal protease, pepsin, elastase, glutaminase, aspartyl proteinase, LysN protease, and ArgC protease;

The guest small molecules with stronger binding affinity with the main group include: mainly one or more of 1,1'-bis(trimethylaminomethyl)ferrocene, dimeric adamantane diammonium ion and the like.

(7) The collected peptide samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) standard-free quantitative analysis technology, sample separation was performed using a C18 chromatographic column, and data was collected using a high-resolution mass spectrometer. The standard-free quantitative analysis technology is a protein quantitative algorithm based on ion current chromatographic peaks. No additional quantitative labels need to be introduced into the protein sample. Only the initial protein amount of the sample needs to be kept consistent. After the peptide samples are collected by mass spectrometry, the mass spectrometry data of the primary spectrum is converted into a three-dimensional map with chromatographic retention time (Retention time), mass-to-charge ratio (m/z), and intensity of the peptide in the primary spectrum as variables, that is, all isotope peak signal intensities of the corresponding mass-to-charge ratio at different retention time points are extracted, and the quantitative information of these peptides is analyzed using the MaxquantLFQ (MaxLFQ) algorithm module to obtain the relative quantitative ratio of the corresponding protein.

Cell surface protein markers related to diseases, cell differentiation, drug resistance mechanisms and other phenotypes were preliminarily identified through differences in LFQ intensity of proteins (proteins with LFQ intensity differences greater than 1.5 between group samples);

At the same time, the peptide fragments of the identified protein are sequenced using the secondary spectrum to perform protein identification.

(8) The mass spectrometry data were analyzed using the software Proteome Discoverer (Thermo Fisher) and Maxquant (https://www.maxquant.org/), and the enriched proteins were qualitatively analyzed. The label-free quantification module in Maxquant was used for quantitative analysis to compare the expression differences of cell surface proteins under different culture and stimulation conditions or between different types of cells, and to preliminarily obtain potential surface proteins related to diseases, cell differentiation, and intercellular communication.

(9) Use search engines such as Uniprot (https://www.uniprot.org/), DAVID Bioinformatics Resources (https://david.ncifcrf.gov/), GORILLA (http://cbl-gorilla.cs.technion.ac.il/), PANTHER Classification System (http://pantherdb.org/), and KEGG (https://www.genome.jp/kegg/) to perform gene ontology annotation and enrichment analysis (GO analysis) and KEGG pathway analysis on the identified proteins. Collect annotation information on molecular functions, biological processes, cellular composition, and KEGG pathways with p values less than or equal to 0.05 or FDR less than or equal to 1%. Analyze the specific localization and functional information of the identified proteins and obtain information on the biological processes and protein pathways in which they participate.

The information obtained can be used to further identify cell surface proteins that can serve as potential disease markers, drug targets, or are important for cell differentiation, molecular transport, and intercellular communication.

The present invention utilizes the characteristics of high binding affinity, specific recognition and controlled release under mild conditions of host-guest interaction, and introduces it into the global in situ analysis of cell surface proteins. A variety of guest-labeled probes with different reaction sites are used to label cell surface proteins in an in situ environment, the cells are lysed, and the enriched material of the immobilized host molecule is put into the cell lysate. The high binding affinity and specific interaction of the host and guest are used to enrich and separate the cell surface proteins labeled by the guest molecule. The enriched protein can be enzymatically hydrolyzed on the material according to the downstream needs, or the enriched protein can be competitively eluted by applying a guest small molecule with a stronger binding affinity with the host group and pre-treated by FASP. Combined with liquid-mass spectrometry technology and label-free quantitative methods, the cell surface proteome is analyzed in situ. This method is of great significance for analyzing intercellular communication, the transport of ions and other molecules, and the transmission of signal cascades, understanding the pathways involved in diseases, immunity, drug resistance, and cell function differentiation, and finding new disease markers and new targets in drug development, thereby providing theoretical support for the occurrence and development of diseases, drug treatment, and inhibition of drug resistance.

The present invention has the following advantages:

(1) The present invention uses host-guest recognition technology as a method for enriching target proteins, overcoming the problems of the biotin-avidin enrichment system that may contaminate the enriched proteins, easily lose low-abundance proteins, and make it difficult to recover the enriched proteins, making the present invention an effective method for in situ analysis of low-abundance cell surface proteomes.

(2) The present invention integrates the host-guest pair enrichment system with the protein labeling technology of multiple reaction sites, which solves the problem of the response preference of the labeling group of a single reaction site to the protein. Under the robust interaction between the host and the guest, it can obtain reproducible and accurate in situ identification of the cell surface proteome with deep coverage.

(3) The present invention combines the enrichment technology based on host-guest interaction with the label-free quantitative proteomics method, which solves the problem that low-abundance cell surface proteins are difficult to accurately identify and quantify, and can be used for in situ research on the expression differences of low-abundance cell surface proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the synthesis process of Fe-Sulfo-NHS ester and Fe-N-NHS ester, wherein a is the synthesis process of Fe-Sulfo-NHS ester and b is the synthesis process of Fe-N-NHS ester.

Figure 2. LTQ molecular weights of Fe-Sulfo-NHS ester and Fe-N-NHS ester, where a is the LTQ molecular weight of Fe-Sulfo-NHS ester and b is the LTQ molecular weight of Fe-N-NHS ester.

Fig. 3. XPS characterization results of Si-CB[7].

Detailed ways

Example 1

(1) Preparation of Silica-CB[7] Main Molecule: 16 mL of deionized water, 2.6 mL of ammonia (28%), and 3.44 mL of tetraethoxysilane were added to 92 mL of anhydrous ethanol in sequence, and the mixture was reacted at room temperature for 16 hours. After the reaction, the mixture was centrifuged at 11000 rpm and the supernatant was discarded to obtain crude silica, which was then ultrasonically washed three times with deionized water and anhydrous ethanol in sequence, and dried overnight to obtain pure silica. The obtained silica was dispersed in 40 mL of anhydrous toluene, 2 mL of 3-(2,3-epoxypropoxy)propyltrimethoxysilane was added, and the mixture was refluxed at 120°C overnight. After the reaction, the mixture was centrifuged at 11000 rpm and the supernatant was discarded to obtain crude epoxy silica, which was then ultrasonically washed three times with acetone and anhydrous ethanol in sequence, and dried overnight to obtain pure epoxy silica. 500 mg of the prepared epoxidized silica and 50 mg of CB[7]-OH were refluxed in 20 mL of acetonitrile at 85° C. for 5 hours, and then 100 μL of triethylamine was added to continue the reaction for 12 hours. The crude product was washed three times with acetonitrile and dried to obtain a pure silicon-CB[7] main material.

(2) Preparation of Sulfo-NHS ester-ferrocene: 2mM ferrocenecarboxylic acid, 2mM N-Boc, 2.4mM HBTU, 4mM DIEA were placed in a 50mL flask with 20mL DMF as solvent, and the reactants were fully dispersed by ultrasound. The oily substance Fe-N-Boc (molecular weight 460.2) was obtained by fully reacting at room temperature for 12 hours. The reaction mixture was purified by semi-preparative liquid phase to obtain pure Fe-N-Boc. The semi-preparative liquid phase separation conditions were: phase A was 0.1% trifluoroacetic acid aqueous solution, phase B was 100% acetonitrile; the gradient was: 0-30min, 30-60% B. The obtained Fe-N-Boc was dissolved in 20mL 5:2 (v/v) dichloromethane:trifluoroacetic acid, reacted at room temperature for 2 hours, and then the solvent was removed by rotary evaporation. 20mL dichloromethane was added and dried by rotary evaporation. This was repeated 3 times to obtain pure Fe-N-NH ₂ (molecular weight 360.2). 0.2mM Fe-N-NH ₂ was mixed with 0.3mM glutaric anhydride, triethylamine was added to adjust the solution to alkalinity (pH = 9), and refluxed in 20mL dichloromethane at 50 degrees for 12h. After the reaction, the dichloromethane was spin-dried and washed three times with dichloromethane to obtain relatively pure Fe-N-COOH (molecular weight 474.1). The prepared 0.1mM Fe-N-COOH was mixed with 0.15mM EDC and 0.15mM Sulfo-NHS, and reacted for 12h under the condition of 20mL DMF as solvent to obtain crude product Fe-Sulfo-NHS ester. The crude product was purified by semi-preparative liquid phase to obtain pure Fe-Sulfo-NHS ester. The semi-preparative liquid phase conditions are: phase A is 0.1% trifluoroacetic acid aqueous solution, phase B is 100% acetonitrile; gradient is: 0-30min, 30-60% B.

(3) Enrichment of plasma membrane proteins of Hela cells using Fe-Sulfo-NHS ester:

5×10 ⁷ Hela cell culture medium was aspirated, washed twice with PBS, and labeled for 5 min on a shaker at 4 degrees with 10 mL of 0.25 mg/mL Fe-Sulfo-NHS ester/PBS solution. The labeling reagent was aspirated, and 10 mL of 100 mM glycine/PBS solution was added. The reaction was quenched in a shaker at 4 degrees for 10 min. After the quenching reagent was aspirated, 10 mL of TBS buffer was added to wash once. RIPA (strong) lysis buffer containing 1% (v/v) protease inhibitor was added at a ratio of 1 mL per 2×10 ⁷ cells. The protein complex was extracted by ultrasonication at 120 W for 2 min. The total protein concentration was measured using a BCA kit. Silica-CB[7] was activated three times with 1 mL of RIPA, added to the protein suspension, enriched at room temperature for 2 h, and the supernatant was removed by centrifugation. 1 mL of 200 mM dithiothreitol was added to the material, and the protein complex sample was denatured and reduced by incubating in a water bath at 56 degrees for 30 min. The supernatant was removed by centrifugation, and the material was washed once with 1mL 50mM ammonium bicarbonate solution; then 1mL 200mM iodoacetamide was added for alkylation reaction in the dark for 30min; the supernatant was removed by centrifugation, and washed several times with 1mL 50mM ammonium bicarbonate solution; finally, 2μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14h; the supernatant was collected by centrifugation at 16000g, which was the peptide sample. The obtained peptide samples were lyophilized, re-dissolved with 0.1% (v/v) formic acid, and data were collected by liquid chromatography-mass spectrometry. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine, and the experimental group and the blank control group were searched without standard quantitative library. The search results were processed by Perseus_1.5.8.5 software to find differential proteins with quantitative intensity ratio greater than 2 and uniquepeptide number greater than or equal to 3, and the protein names, cell distribution and other information were annotated on the Uniprot website and DAVID website.

Example 2

(1) The operation is as shown in step (1) in Example 1.

(2) Preparation of ferrocene with NHS ester: 2mM ferrocenecarboxylic acid, 2mM N-Boc, 2.4mM HBTU, 4mM DIEA were placed in a 50mL flask with 20mL DMF as solvent, and the reactants were fully dispersed by ultrasound. The oily substance Fe-N-Boc (molecular weight 460.2) was obtained by fully reacting at room temperature for 12 hours. The reaction mixture was purified by semi-preparative liquid phase to obtain pure Fe-N-Boc. The semi-preparative liquid phase separation conditions were: phase A was 0.1% trifluoroacetic acid aqueous solution, phase B was 100% acetonitrile; the gradient was: 0-30min. The obtained Fe-N-Boc was dissolved in 20mL 5:2 (v/v) dichloromethane:trifluoroacetic acid, reacted at room temperature for 2 hours, and then the solvent was removed by rotary evaporation. 20mL dichloromethane was added and dried by rotary evaporation. This was repeated 3 times to obtain pure Fe-N-NH ₂ (molecular weight 360.2). 0.2mMFe-N-NH ₂ was mixed with 0.3mM glutaric anhydride, triethylamine was added to adjust the solution to alkalinity (pH = 9), and refluxed in 20mL dichloromethane at 50 degrees for 12h. After the reaction, the dichloromethane was spin-dried and washed three times with dichloromethane to obtain relatively pure Fe-N-COOH (molecular weight 474.1). The prepared 0.1mM Fe-N-COOH was mixed with 0.15mM EDC and 0.15mM NHS, and reacted for 12h under the condition of 20mL DMF as solvent to obtain crude product Fe-NHS ester. The crude product was purified by semi-preparative liquid phase to obtain pure Fe-NHS ester. The semi-preparative liquid phase conditions are: phase A is 0.1% trifluoroacetic acid aqueous solution, phase B is 100% acetonitrile; gradient is: 0-30min, 30-60% B.

(3) Enrichment of plasma membrane proteins of Hela cells using Fe-N-NHS ester:

5×10 ⁷ Hela cell culture medium was aspirated, washed twice with PBS, and labeled for 5 min on a shaker at 4 degrees with 10 mL of 0.25 mg/mL Fe-N-NHS ester/PBS solution. The labeling reagent was aspirated, and 10 mL of 100 mM glycine/PBS solution was added. The reaction was quenched in a shaker at 4 degrees for 10 min. After the quenching reagent was aspirated, 10 mL of TBS buffer was added to wash once. RIPA (strong) lysis buffer containing 1% (v/v) protease inhibitor was added at a ratio of 1 mL per 2×10 ⁷ cells. The protein complex was extracted by ultrasonication at 120 W for 2 min. The total protein concentration was measured using a BCA kit. Silica-CB[7] was activated three times with 1 mL of RIPA, added to the protein suspension, enriched at room temperature for 2 h, and the supernatant was removed by centrifugation. 1 mL of 200 mM dithiothreitol was added to the material, and the protein complex sample was denatured and reduced by water bath at 56 degrees for 30 min. The supernatant was removed by centrifugation, and the material was washed once with 1mL 50mM ammonium bicarbonate solution; then 1mL 200mM iodoacetamide was added for alkylation reaction in the dark for 30min; the supernatant was removed by centrifugation, and washed several times with 1mL 50mM ammonium bicarbonate solution; finally, 2μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14h; the supernatant was collected by centrifugation at 16000g, which was the peptide sample. The obtained peptide samples were lyophilized, re-dissolved with 0.1% (v/v) formic acid, and data were collected by liquid chromatography-mass spectrometry. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine, and the experimental group and the blank control group were searched without standard quantitative library. The library search results were processed by Perseus_1.5.8.5 software to find differential proteins with quantitative intensity ratio greater than 2 and unique peptide number greater than or equal to 3, and the protein name, cell distribution and other information were annotated on the Uniprot website and DAVID website.

Example 3

(1) The operation is as shown in steps (1) and (2) in Example 1.

(2) Fe-Sulfo-NHS ester was used to enrich and analyze the differentially expressed proteins in the cell surface proteome of common hematological cell lines K562, U937, HL-60, KG-1, and Molm-13:

The cell culture medium of 5×10 ⁷ K562, U937, HL-60, KG-1, and Molm-13 cells was aspirated, washed twice with PBS, and labeled for 5 min on a shaker at 4 degrees with 10 mL of 0.25 mg/mL Fe-Sulfo-NHS ester/PBS solution. The labeling reagent was aspirated, and 10 mL of 100 mM glycine/PBS solution was added. The reaction was quenched in a shaker at 4 degrees for 10 min. After the quenching reagent was aspirated, 10 mL of TBS buffer was added to wash once. RIPA (strong) lysis buffer containing 1% (v/v) protease inhibitor was added at a ratio of 1 mL per 2×10 ⁷ cells. The protein complex was extracted by ultrasonication at 120 W for 2 min. The total protein concentration was measured using a BCA kit. Silica-CB[7] was activated three times with 1 mL of RIPA, added to the protein suspension, enriched at room temperature for 2 h, and centrifuged to remove the supernatant. Add 1mL of 200mM dithiothreitol to the material, and place in a water bath at 56 degrees for 30min to denature and reduce the protein complex sample. Remove the supernatant by centrifugation, and wash the material once with 1mL of 50mM ammonium bicarbonate solution; then add 1mL of 200mM iodoacetamide for alkylation reaction in the dark for 30min; remove the supernatant by centrifugation, and wash it several times with 1mL of 50mM ammonium bicarbonate solution; finally, add 2μg of trypsin (trypsin: protein complex mass ratio = 1:25) for enzymatic digestion for 14h; collect the supernatant by centrifugation at 16000g, which is the peptide sample. The obtained peptide sample was lyophilized, re-dissolved with 0.1% (v/v) formic acid, and data was collected by liquid chromatography-mass spectrometry. All data collected by mass spectrometry were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine. Standard-free quantitative library search was performed between experimental groups. The expression abundance of proteins in the experimental groups was absolutely quantified using iBAQ values. The library search results were processed using Perseus_1.5.8.5 software to find differential proteins with relative quantitative intensity ratios greater than 2 and unique peptide numbers greater than or equal to 3, as well as the expression abundance of these proteins in their respective groups. The protein names, cell distribution and other information were annotated on the Uniprot website and DAVID website. Cell surface proteins that are commonly highly expressed in common cell lines in hematological diseases and cell surface proteins that are differentially expressed between cell lines were found.

Example 4

(1) The operation is as shown in steps (1) and (2) in Example 1.

(2) Fe-Sulfo-NHS ester was used to enrich the cell surface proteins of the K562-TRE3G-ETV6-MECOM cell model, and the expression changes of the K562 cell surface proteome caused by the ETV6-MECOM gene were analyzed:

The culture medium of 5×10 ⁷ K562-TRE3G-ETV6-MECOM cells induced with DOX (experimental group) and without DOX (control group 1) and empty cells induced with DOX (control group 2) and without DOX (control group 3) was aspirated, washed twice with PBS, and labeled for 5 minutes on a shaker at 4 degrees with 10 mL of 0.25 mg/mL Fe-Sulfo-NHS ester/PBS solution. The labeling reagent was aspirated, and 10 mL of 100 mM glycine/PBS solution was added, and the reaction was quenched on a shaker at 4 degrees for 10 minutes. After the quenching reagent was aspirated, 10 mL of TBS buffer was added to wash once. RIPA (strong) lysis buffer containing 1% (v/v) protease inhibitor was added at a ratio of 1 mL per 2×10 ⁷ cells, and the protein complex was extracted by ultrasonication at 120 W for 2 minutes. The total protein concentration was measured using a BCA kit. Silica-CB[7] was activated three times with 1 mL RIPA, added to the protein suspension, enriched at room temperature for 2 h, and centrifuged to remove the supernatant. 1 mL of 200 mM dithiothreitol was added to the material, and the protein complex sample was denatured and reduced in a water bath at 56 degrees for 30 min. The supernatant was removed by centrifugation, and the material was washed once with 1 mL 50 mM ammonium bicarbonate solution; then 1 mL 200 mM iodoacetamide was added for alkylation reaction in the dark for 30 min; the supernatant was removed by centrifugation, and washed several times with 1 mL 50 mM ammonium bicarbonate solution; finally, 2 μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14 h; the supernatant was collected by centrifugation at 16000 g, which was the peptide sample. The obtained peptide sample was lyophilized, reconstituted with 0.1% (v/v) formic acid, and data was collected by liquid chromatography-mass spectrometry. All the data collected by mass spectrometry were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine. The experimental group and the control group were searched without label quantitative library. The search results were processed using Perseus_1.5.8.5 software to find differential proteins with quantitative intensity ratio greater than 2 and unique peptide number greater than or equal to 3. The protein name, cell distribution and other information were annotated on the Uniprot website and DAVID website. Differential cell surface proteins caused by the influence of the fusion gene ETV6-MECOM were found.

Claims (9)

1. An in situ analysis method of cell surface proteome based on host-guest interaction and no calibration, characterized in that: the method comprises the following steps:

(1) Modifying a host group on a matrix carrier to prepare a host enrichment separation material specifically recognizing a guest group;

(2) Connecting a guest group with a labeling group to prepare a bifunctional guest labeling probe molecule which has an impermeable membrane and can be enriched;

(3) Incubating the prepared object marked probe molecule with cells, and in-situ covalent marking plasma membrane protein and cell surface protein on the surfaces of the cells by the probe;

(4) Disrupting the cells in step (3), extracting a protein mixture from the cells, incubating the protein mixture with the host enrichment and separation material prepared in step (1), and selectively capturing the proteins labeled with the guest-labeled probes from the heterogeneous protein mixture using high affinity interactions between the host and the guest;

(5) Processing the protein enriched in the step (4), and collecting a peptide fragment sample;

(6) And (3) combining a liquid chromatography-mass spectrometry technology and no calibration quantity, and carrying out in-situ quantitative analysis on the peptide fragment sample in the step (5).

2. The method of in situ analysis of cell surface proteomes of claim 1, wherein: the host group described in step (1) includes: one or more of cucurbituril [ n ] uril (n=5-8, 10, 14), cyclodextrin, calixarene, and column [ n ] arene (n=5, 6);

The matrix carrier comprises: one or more than two of agarose, high molecular polymer nano particles, mesoporous silica materials, carbon nanotubes, ferroferric oxide magnetic spheres and metal organic framework materials;

The modification mode comprises one or two of physical modification or chemical modification, wherein the physical modification comprises one of coprecipitation and surface adsorption; the chemical modification comprises one or more than two of EDC/NHS coupling, addition reaction of epoxy group and hydroxyl group, amino group, carboxyl group and the like, covalent bonding of sulfhydryl group and maleimide, alpha-haloacetyl or gold.

3. The method of in situ analysis of cell surface proteomes of claim 1, wherein: the guest group described in step (2) includes: one or more of ferrocene, adamantane, dimer adamantane, polyhedral boron clusters, ammonium salts, imidazolium salts and pyridinium salts;

The labeling group comprises: succinimide groups reactive with amino groups on proteins, halogenated aromatic hydrocarbons or imidoesters, acylphloroglucinols, phthalaldehyde, squarates, sulfonyl fluorides, polycarbonyl, aldehyde ketone derivatives; or a maleimide group, 2-mercaptopyridine, thiosulfonate, haloacetyl or pyridyl disulfide group that can react with a thiol group on a protein; or carbodiimide or isocyanate that can react with carboxyl groups on proteins; or a hydrazide group which can react with a sugar chain on a protein; or a non-specifically reactive group that can react with an amino acid residue in a protein, a benzophenone group, a phenyl azide group, a bisaziridine, or a 2-aryl-5-carboxytetrazole; or a phenol group that can react with a phenol group on a protein.

4. The method of in situ analysis of cell surface proteomes of claim 1, wherein: the guest tagged probe molecules co-incubate with the cells in step (3) as follows: the probes were incubated with cells in serum-free cell culture medium at 4-37℃for 5 min-12 h under 5% CO ₂ and cells were collected.

5. The method of in situ analysis of cell surface proteomes of claim 1, wherein: the cell disruption in the step (4) and the process of extracting all proteins from the cells comprises the following steps: adding cell lysate containing protease inhibitor cocktail into cells, ultrasonically crushing cells, centrifuging 10000-16000g for 5-30 min, and taking out supernatant sample;

The cell lysate comprises: 1-butyl-3-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole tetrafluoroacetate, 1-butyl-3-methylimidazole trifluoromesylate, bromo-1-butyl-3-methylimidazole tetrafluoroborate, chloro-1-butyl-3-methyl-imidazole, chloro-1-ethyl-3-methylimidazole, chloro-1-octyl-3-methylimidazole, chloro-1-dodecyl-3-methylimidazole, chloro-1-allyl-3-methylimidazole, 1-aminopropyl-3-methylimidazole tetrafluoroborate, 1-hydroxyethyl-3-methylimidazole tetrafluoroborate, chloro-N-butylpyridine, N-butylpyridine tetrafluoroborate, urea, guanidine hydrochloride, sodium lauryl sulfate;

The ratio of cells to cell lysate was: 200-600. Mu.l of cell lysate was added to 3X 10 ⁶ cells;

the protease inhibitor cocktail in the cell lysate accounts for 0.5-5% (v/v);

the ultrasonic power is 80-160W, and the ultrasonic time is 1-5 minutes.

6. The method of in situ analysis of cell surface proteomes of claim 1, wherein: the process of incubating the protein mixture with the host enrichment material in step (4) comprises: incubating the protein mixture with the enrichment material at 4-37 ℃ for 2-16 hours;

the ratio of enrichment material to total protein in the protein mixture is: 1mg of whole protein is added to 0.1-10mg of enrichment material.

7. The method of in situ analysis of cell surface proteomes of claim 1, wherein: the method for processing the enriched protein in the step (5) comprises the following steps: the method comprises the steps of (1) carrying out enzymolysis treatment on enriched proteins directly on an enrichment material, and collecting peptide fragment samples;

Or performing competitive elution on the protein enriched on the enrichment material by using a guest small molecule with stronger binding affinity with a host group, and then performing sample pretreatment by FASP (based on a filter membrane auxiliary sample preparation method), and collecting a peptide fragment sample;

Wherein the protease used for enzymolysis comprises one or more of trypsin, endolys-enzyme, chymotrypsin, lysine-arginine N-terminal protease, pepsin, elastase, endoglutaminase, aspartyl-endoprotease, lysN-protease and ArgC protease;

the guest small molecules with higher binding affinity to the host group include: mainly comprises one or more than two of 1,1' -bis (trimethyl aminomethyl) ferrocene, dimer adamantane diammonium ion and the like.

8. The method of in situ analysis of cell surface proteomes of claim 1, wherein:

The non-calibration analysis technology is a protein quantitative algorithm based on ion flow chromatographic peaks, no extra quantitative labels are required to be introduced into a protein sample, the initial protein amount of the sample is kept consistent, after a peptide fragment sample is acquired by mass spectrometry, mass spectrum data of a primary spectrogram is converted into a three-dimensional spectrogram taking chromatographic Retention time (Retention time), mass-to-charge ratio (m/z) and Intensity (Intensity) of a peptide fragment in the primary spectrogram as variables, namely, all isotope peak signal intensities of the corresponding mass-to-charge ratio at different Retention time points are extracted, and quantitative information of the peptide fragments is analyzed by utilizing MaxquantLFQ (MaxLFQ) algorithm modules, so that the relative quantitative ratio of the corresponding protein is obtained;

Preliminary identification of cell surface protein markers associated with phenotypes such as disease, cell differentiation, drug resistance mechanisms, etc. by LFQ intensity differences of proteins (proteins with LFQ intensity differences greater than 1.5 in samples between groups);

And simultaneously, determining the sequence of the peptide fragment of the identified protein by using a secondary spectrogram so as to identify the protein.

9. The method of in situ analysis of cell surface proteomes of claim 1, wherein: the cell comprises: tumor cells (sensitive strain, drug-resistant strain), immune cells, stem cells, and healthy cells.