CN118169263A - Cell surface proteome in-situ analysis method based on interaction of host and guest and without calibration - Google Patents
Cell surface proteome in-situ analysis method based on interaction of host and guest and without calibration Download PDFInfo
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- CN118169263A CN118169263A CN202211602865.6A CN202211602865A CN118169263A CN 118169263 A CN118169263 A CN 118169263A CN 202211602865 A CN202211602865 A CN 202211602865A CN 118169263 A CN118169263 A CN 118169263A
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- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- PWEBUXCTKOWPCW-UHFFFAOYSA-L squarate Chemical compound [O-]C1=C([O-])C(=O)C1=O PWEBUXCTKOWPCW-UHFFFAOYSA-L 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical class [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- HQYALQRYBUJWDH-UHFFFAOYSA-N trimethoxy(propyl)silane Chemical compound CCC[Si](OC)(OC)OC HQYALQRYBUJWDH-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
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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
Technical Field
The invention relates to a novel in-situ analysis method of a cell surface proteome, and belongs to the technical field of biological analysis.
Background
Cell surface proteins refer to proteins exposed on the cell surface, including and acting as the first barrier to living cells, and are responsible for performing many critical functions including intercellular interactions, signal transduction, and molecular trafficking, among others. The difference of cell surface protein expression not only distinguishes important markers of different types of cells, but also is the basis of phenotype and function differences between normal cells and diseased cells, so that nearly 2/3 of drug target proteins belong to cell surface proteins. Identification of cell surface proteomes and study of the properties of these proteins are key steps in understanding basic biological processes and in finding new targets of action in drug development (Expert Review of Proteomics,2010,7 (1), 141-154; J.Sep. Sci.,2020,43,292-312).
The global profile of the cell surface proteome has been a major challenge for proteomics because of the low abundance, high hydrophobicity and high heterogeneity that limit the separation and extraction efficiency of cell surface proteins. In recent years, chemical proteomics has emerged to provide a new tool for the identification of cell surface proteins by chemically labeling surface-exposed protein or side chain groups of sugar chains (e.g., primary amine, carboxyl, glycan side chains), followed by separation of the labeled surface proteins by affinity purification, such as sugar capture, metabolic labeling, and cell surface biochemistry. The main advantage of this type of technology is that the chemical labelling reagents can be custom designed according to the target biological structure or requirements, and only a small initial cell amount is required to obtain high purity cell surface proteins. At present, cell surface biotinylation is most commonly used, namely, small molecule biotin is modified on cell surface proteins, and a receptor streptavidin is used for specifically extracting labeled proteins, but a biotin-avidin system has the risk of polluting enriched protein samples, firstly, interference of endogenous biotinylation proteins existing in cells masks low-abundance proteins, and detection sensitivity is reduced; secondly, streptavidin itself is also protein and can be hydrolyzed, which may become high abundance pollution protein in the sample; in addition, streptavidin is also prone to denaturation under high temperature or harsh wash conditions, thereby freeing the matrix material, leading to loss of enriched protein, hampering identification of low abundance cell surface proteins (Nature Chemistry,2011,3,154-159). Therefore, the method uses a chemically synthesized host-guest interaction system with smaller size and higher physical and chemical stability to analyze the cell surface protein, and obtains stable and reproducible surface proteome information with higher purity and deeper coverage.
Host guest interaction refers to a class of supermolecular chemistry in which host and guest molecules interact through non-covalent bonds to form inclusion complexes. The host molecules currently developed mainly include crown ethers, cyclodextrins, cucurbiturils, pillar and calixarenes, which specifically recognize and bind with high affinity such as adamantane, ferrocene, carborane and its derivatives, and cation or anion rich guest molecules, mainly through non-covalent interactions such as hydrophobic interactions, hydrogen bonds, electrostatic interactions, pi-pi stacking, and the like, and this binding is reversible. Based on the above characteristics, host-guest interactions have been applied in various fields such as biosensors, catalytic regulation, development of underwater adhesives, protein separation, drug delivery, and the like.
In cell surface protein separation, the host-guest recognition technology is not easy to be influenced by enzymolysis or severe washing conditions to cause the loss of captured proteins due to stable chemical structures, and can selectively release the captured proteins by applying competing guest molecules so as to facilitate mass spectrum identification. In addition, due to the expandability of chemical synthesis, the host-guest recognition molecules can be modified according to different analysis purposes while being low in cost and easy to obtain, and the specificity and sensitivity of the method are improved. Through combination with analysis technologies such as western blotting and fluorescence imaging, the method has the advantages that the method has higher enrichment efficiency and lower nonspecific adsorption protein interference compared with a common biotin-avidin system, can release enriched protein under mild conditions, and can realize accurate imaging of the protein (Nature Chemistry,2011,3,154-159;Nature Communications,2018,9,1712-1722). But this technique has not been combined with mass spectrometry based proteome quantitative analysis to globally analyze the cell surface proteome.
The non-calibration technology is to carry out mass spectrometry on the protein enzymolysis peptide fragments by a liquid chromatography-mass spectrometry technology, expensive stable isotope labels are not required to be used as internal standards, and only the signal intensities of the corresponding peptide fragments in different samples are required to be compared, so that the relative quantification of the proteins corresponding to the peptide fragments is carried out. The nonstandard quantity can be used for simultaneously carrying out quantitative analysis on a plurality of groups of samples, so that the experimental flux is improved.
Therefore, in order to resolve cell surface proteomes with great precision, a host-guest recognition technique with high affinity and dynamic binding can be combined with mass spectrometry-based proteome non-quantitative analysis.
In the patent, aiming at the problems that the existing biotin-avidin labeling system possibly pollutes enriched proteins, is easy to lose low-abundance proteins, is difficult to recover the enriched proteins and the like, a host-guest recognition technology with the characteristics of high binding affinity, good physical and chemical stability, low biological background, mild release of labeled proteins and the like is utilized, and the host-guest recognition technology is combined with a mass spectrometry quantitative analysis method, so that a cell surface proteome in-situ analysis method based on host-guest interaction and no calibration amount is developed, and the deep and accurate analysis of cell surface proteins is realized.
Disclosure of Invention
The invention aims to provide an in-situ analysis method of cell surface proteome based on interaction of a host and a guest and without a calibrated amount, and global analysis of the cell surface proteome under a cell in-situ environment is realized by the method.
An in-situ analysis method of cell surface proteome based on host-guest interaction and no calibration amount specifically comprises the following steps:
(1) Preparing a main body enrichment material, wherein main body groups are covalently bonded on a matrix material, and the main body groups consist of one or more than two of crown ether, cucurbit [ n ] urils (n=5-8, 10, 14), cyclodextrin, calixarene and column [ n ] arene (n=5, 6); the matrix material is mainly one or more than two of agarose, high polymer nano particles, mesoporous silica material, carbon nano tube, ferroferric oxide magnetic sphere, metal organic framework material and the like.
Constructing a host enrichment material from the two aspects of improving the binding affinity with a guest molecule, reducing the nonspecific adsorption and the potential pollution to a protein sample, and optimizing the surface hydrophilicity and the physical and chemical stability (such as acid and alkali resistance, detergent, high temperature and the like) of the material to obtain a matrix material with low nonspecific adsorption and small pollution to the sample; the enrichment efficiency is improved by adjusting the bonding amount of the main body group and the type of the main body group.
(2) Preparing a guest labeling probe, and connecting a guest group with a labeling group; the guest groups may be: ferrocene, adamantane, dimer adamantane, polyhedral boron clusters, ammonium salts, imidazolium salts and pyridinium salts, and derivatives thereof; the labeling group includes: one or more of sulfosuccinimidyl ester, N-succinimidyl ester, acylphloroglucinol, phthalaldehyde, squarate, sulfonyl fluoride, polycarbonyl, aldehyde ketone, halogenated aromatic hydrocarbon or imido ester, maleimide, 2-mercaptopyridine, thiosulfate, halogenated acetyl or pyridyl disulfide, carbodiimide or isocyanate, hydrazide, phenol group, benzophenone group, phenyl azide group, trifluoromethyl biaziridine, biaziridine or 2-aryl-5-carboxytetrazole, and the like.
The guest labeling probe is designed based on the principles of good impermeability, biocompatibility, rapid and efficient reactivity, reaction site complementarity and high binding property with host molecules, and the probe is difficult to permeate a membrane by introducing a negatively charged group on a labeling group and using a connecting arm with strong hydrophilicity, so that the guest labeling probe mainly reacts with cell surface proteins; selecting a light crosslinking marking group which has different reaction sites and better selectivity or a site nonspecific light crosslinking marking group, and introducing a group which can generate noncovalent action with a protein amino acid side chain to meet the requirements of good biocompatibility, high reaction efficiency and site complementation; optimizing the types of the guest groups and the side chain groups with different chargeability to obtain the guest labeling probe which can be efficiently and specifically identified by the host enrichment material.
(3) The guest labeling probe labels a cell surface protein. The guest-labeled probe and the cells were incubated in serum-free medium at 4℃or 37℃under 5% CO 2 for 5 minutes to 12 hours, and the cells were collected. Because of poor membrane permeability, the probe is mainly accumulated outside the cell membrane or between cells, and then the labeling group is modified to the cell surface protein by covalent reaction with the side chain of nucleophilic amino acid or by formation of free radical under ultraviolet light driving into X-H (x=c, N, O) bond on the protein.
(4) Disrupting cells, adding cell lysate containing 0.5-5% (v/v) protease inhibitor cocktail to the collected cells, adding 200-600 μl lysate per 3×10 6 cells, sonicating cells, centrifuging 10000-16000g for 5-30 min, and taking out supernatant to obtain protein mixed 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.
(5) Enriching cell surface proteins. Mixing the obtained protein mixed sample with main enrichment material at a ratio of 1mg total protein to 0.1-10mg enrichment material, and incubating at 4-37deg.C for 2-16 hr. Allowing the host enrichment material to fully contact with the guest molecules on the proteins and achieve high binding affinity binding, thereby enriching the cell surface proteins modified with the guest molecules on the material; centrifugation at 1000-16000g for 5-10 min, discarding the supernatant and separating cell surface proteins from other intracellular proteins.
(6) Treatment of the enriched protein
The method comprises the following steps of directly carrying out enzymolysis treatment on the enriched protein on an enriched material: dispersing the material enriched with protein in cell lysis, adding dithiothreitol or tri (2-carboxyethyl) phosphine, performing denaturation and reduction at 25-37 ℃ for 5 min-2 hours, centrifuging at 1000-16000g for 5-10min, and discarding the supernatant; the enrichment material after the denaturation of the disperse protein of the cell lysate is reused, the iodoacetamide is utilized to carry out light-proof alkylation for 20 to 40 minutes at 25 ℃, and the supernatant is centrifuged and discarded; fully dispersing the enrichment material after protein alkylation by using 50mM ammonium bicarbonate, adding protease, carrying out enzymolysis for 12-16 hours according to the ratio of protease to protein complex of 1:20-1:50 (w/w), centrifuging for 5-10 minutes by 1000-16000g, and collecting the supernatant containing the peptide fragment sample;
Or compete for elution of proteins from the enrichment material and sample pretreatment with FASP. Dispersing the material enriched with protein in cell lysate, adding guest small molecules (1, 1' -bis (trimethylammoniomethyl) ferrocene) with stronger binding affinity with host groups, incubating at 25-95 ℃ for 5 min-2 h, centrifuging and collecting supernatant containing protein; adding dithiothreitol or tris (2-carboxyethyl) phosphine into the eluted protein solution, and carrying out high-temperature denaturation and reduction, wherein the high-temperature denaturation temperature is 37-95 ℃ and the time is 5 minutes-2 hours; transferring the sample to a 3k-10k Da filter membrane, and carrying out light-proof alkylation by using iodoacetamide for 20-40 minutes; protease was added, protease 1:2000-1:5000 (w/w): and (3) carrying out enzymolysis for 12-16 hours according to the proportion of total protein, and centrifuging to collect peptide fragment samples.
Wherein the protease comprises one or more of trypsin, lys-endoprotease, chymotrypsin, lysine-arginine N-terminal protease, pepsin, elastase, glutamine endoprotease, aspartyl endoprotease, lysN protease, 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.
(7) And analyzing the collected peptide sample by adopting a liquid chromatography-mass spectrometry non-calibration analysis technology, separating the sample by using a C18 chromatographic column, and collecting data by using a high-resolution mass spectrum. The non-calibration analysis technology is a protein quantitative algorithm based on ion flow chromatographic peaks, and is characterized in that no extra quantitative label is required to be introduced into a protein sample, only 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 are converted into a three-dimensional spectrogram taking chromatographic Retention time (Retention time), mass-to-charge ratio (m/z) and strength (integrity) of a peptide fragment in the primary spectrogram as variables, namely, all isotope peak signal intensities of corresponding mass-to-charge ratios 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 corresponding proteins 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.
(8) The plasma spectrum data are analyzed by software Proteome Discoverer (Thermo Fisher) and Maxquant (https:// www.maxquant.org /), the enriched proteins are qualitatively analyzed, and quantitative analysis is performed by using a Label-free quantification module in Maxquant, and the expression difference of the cell surface proteins under different culture and stimulation conditions or between different cell types is compared, so that potential surface proteins related to diseases, cell differentiation, cell-cell communication and the like are primarily obtained.
(9) Carrying out gene ontology annotation and enrichment analysis (GO analysis) and KEGG channel analysis on the identified proteins by using 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/)、KEGG(https://www.genome.jp/kegg/) and other search engines, collecting annotation information of molecular functions, biological processes, cell compositions and KEGG channels with p value less than or equal to 0.05 or FDR less than or equal to 1%, analyzing specific positioning and functional information of the identified proteins, and obtaining the biological processes and protein channel information participated in by the identified proteins.
Further, through the obtained information, the cell surface protein which can be used as a potential disease marker, a drug target or has important significance for cell differentiation, molecular transport and intercellular communication is determined.
The invention utilizes the characteristics of high binding affinity, specific recognition and controllable release of the interaction of a host and a guest under mild conditions to introduce the host and guest into the global in-situ analysis of cell surface proteins. The method comprises the steps of respectively marking cell surface proteins in an in-situ environment by utilizing a plurality of guest labeling probes with different reaction sites, lysing cells, adding an enrichment material for immobilizing host molecules into a cell lysate, enriching and separating the cell surface proteins marked by guest molecules by utilizing high binding affinity and specific interaction of the host and the guest, carrying out enzymolysis on the enriched proteins on the materials according to downstream requirements or carrying out competitive elution on the enriched proteins by applying guest small molecules with stronger binding affinity with the host groups, carrying out sample pretreatment by utilizing FASP, and carrying out in-situ analysis on cell surface proteomes by utilizing a combined liquid chromatography-mass spectrometry technology and a nonstandard quantitative method. 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 new action targets in drug development, and further providing theoretical support for the occurrence and development of diseases, drug treatment and drug resistance inhibition.
The invention has the following advantages:
(1) The invention uses host-guest recognition technology as the enrichment method of target protein, which overcomes the problems that the biotin-avidin enrichment system may pollute the enriched protein, low-abundance protein is easy to lose, the enriched protein is difficult to recycle, and the like, so that the invention becomes an effective method for in-situ analysis of low-abundance cell surface proteome.
(2) According to the invention, the host and the guest are fused with the protein marking technology of the enrichment system and a plurality of reaction sites, so that the problem of reaction preference of a single reaction site marking group to proteins is solved, and in-situ identification of the cell surface proteome with accurate reproduction and deep coverage can be obtained under the steady interaction of the host and the guest.
(3) The invention combines the enrichment technology based on the interaction of the host and the guest with the method of no-calibration amount of proteome, solves the problem that the protein on the surface of the low-abundance cell is difficult to be accurately, qualitatively and quantitatively analyzed, and can be used for in-situ research of the expression difference of the protein on the surface of the low-abundance cell.
Drawings
FIG. 1 shows the synthetic schemes of Fe-Sulfo-NHS ester and Fe-N-NHS ester, wherein a is the synthetic scheme of Fe-Sulfo-NHS ester, and b is the synthetic scheme of Fe-N-NHS ester.
FIG. 2 shows the LTQ characteristic molecular weight of Fe-Sulfo-NHS ester and Fe-N-NHS ester, wherein a is the LTQ characteristic molecular weight of Fe-Sulfo-NHS ester and b is the LTQ characteristic molecular weight of Fe-N-NHS ester.
FIG. 3 XPS characterization results of silicon-based-CB [7 ].
Detailed Description
Example 1
(1) Preparation of a silicon-based-CB [7] host molecule: to 92mL of absolute ethanol, 16mL of deionized water, 2.6mL of ammonia (28%), 3.44mL of tetraethoxysilane were added in this order, and the mixture was reacted at room temperature for 16 hours. After the reaction is finished, the supernatant is centrifuged at 11000rpm to obtain crude product silicon dioxide, deionized water and absolute ethyl alcohol are sequentially used for ultrasonic washing for three times, and the pure silicon dioxide is obtained after drying overnight. The prepared silica was dispersed in 40mL of anhydrous toluene, 2mL of 3- (2, 3-glycidoxy) propyltrimethoxysilane was added, and the mixture was refluxed overnight at 120 ℃. Centrifuging at 11000rpm after the reaction is finished, discarding the supernatant to obtain crude epoxy silicon dioxide, sequentially ultrasonic washing the crude product with acetone and absolute ethyl alcohol for three times, and drying overnight to obtain pure epoxy silicon dioxide. The prepared 500mg of epoxidized silicon dioxide and 50mg of CB 7-OH are reflux reacted in 20mL of acetonitrile at 85 ℃ for 5 hours, then 100 mu L of triethylamine is added for continuous reaction for 12 hours, and the obtained crude product is washed by acetonitrile for 3 times and dried to obtain the pure silicon-based-CB 7 main material.
(2) Preparation of Sulfo-NHS ester-ferrocene: 2mM ferrocenecarboxylic acid, 2mM N-Boc, 2.4mM HBTU and 4mM DIEA are put into a 50mL flask with 20 mM LDMF as a solvent, the reactants are fully dispersed by ultrasound, the reaction is fully carried out for 12 hours at room temperature to obtain oily Fe-N-Boc (molecular weight is 460.2), and the mixture obtained by the reaction is purified by semi-prepared liquid phase to obtain pure Fe-N-Boc. The semi-preparation liquid phase separation conditions are as follows: phase a is 0.1% aqueous trifluoroacetic acid and phase B is 100% acetonitrile; the gradient is: 0-30min,30-60% B. The Fe-N-Boc prepared was dissolved in 20mL 5:2 (v/v) dichloromethane: after reacting in trifluoroacetic acid for 2 hours at room temperature, the solvent is removed by rotary evaporation, and then 20mL of dichloromethane is added for rotary drying, and the reaction is repeated for 3 times, thus obtaining pure Fe-N-NH 2 (with the molecular weight of 360.2). 0.2mM Fe-N-NH 2 was mixed with 0.3mM glutaric anhydride, triethylamine was added to make the solution basic (pH=9), 50℃reflux was carried out in 20mL of methylene chloride for 12h, the methylene chloride was dried by spinning after the reaction was completed, and the methylene chloride was washed three times to obtain purer 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 12 hours under the condition of 20Ml DMF as a solvent to obtain a crude product Fe-Sulfo-NHS ester. The crude product is purified by semi-preparative liquid phase to obtain pure Fe-Sulfo-NHS ester. The semi-preparation liquid phase conditions are as follows: phase a is 0.1% aqueous trifluoroacetic acid and phase B is 100% acetonitrile; the gradient is: 0-30min,30-60% B.
(3) Enrichment of HeLa cells with Fe-Sulfo-NHS ester plasma membrane protein:
HeLa cell culture medium, 5X 10 7, was blotted, washed twice with PBS, and 10mL of 0.25mg/mL Fe-Sulfo-NHS ester/PBS solution was added and labeled on a 4 degree shaker for 5min. The labeled reagent was blotted, 10mL of 100mM glycine/PBS solution was added, the mixture was incubated in a 4℃shaker for 10min to quench the reaction, and after the quenching reagent was blotted, 10mL TBS buffer was added and washed 1 time. RIPA (strong) lysate containing 1% (v/v) protease inhibitor is added at a ratio of 1mL per 2×10 7 cells, and sonicated at 120W power for 2min to extract protein complexes. Total protein concentration was measured using BCA kit. silicon-based-CB [7] is activated three times with 1mL RIPA, added to the protein suspension, enriched for 2h at room temperature, and the supernatant removed by centrifugation. To the material, 1mL of 200mM dithiothreitol was added, and the denaturation reduction of the protein complex sample was performed in a 56-degree water bath for 30 min. The supernatant was removed by centrifugation and the primary material was washed with 1mL of 50mM ammonium bicarbonate solution; 1mL 200mM iodoacetamide is added to carry out alkylation reaction for 30min in dark place; the supernatant was removed by centrifugation and washed multiple times with 1mL of 50mM ammonium bicarbonate solution; finally adding 2 mug trypsin (trypsin: protein complex mass ratio=1:25) for enzymolysis for 14h; the supernatant, i.e., the peptide fragment sample, was collected by centrifugation at 16000 g. The peptide samples obtained were lyophilized and reconstituted with 0.1% (v/v) formic acid and data were collected using liquid chromatography-mass spectrometry. All mass spectrum collected data are searched by adopting MaxQuant-1.6.5.0 with built-in Andromeda search engines, an experimental group and a blank control group perform nonstandard quantitative library searching, data processing is performed on library searching results by Perseus-1.5.8.5 software, differential proteins with quantitative intensity ratio more than 2 and number of Unique peptides more than or equal to 3 are found, and information such as names, cell distribution and the like of the proteins are annotated in Uniprot websites and DAVID websites.
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 and 4mM DIEA are put into a 50mL flask with 20 mM LDMF as a solvent, the reactants are fully dispersed by ultrasound, the reaction is fully carried out for 12 hours at room temperature to obtain oily Fe-N-Boc (molecular weight is 460.2), and the mixture obtained by the reaction is purified by semi-prepared liquid phase to obtain pure Fe-N-Boc. The semi-preparation liquid phase separation conditions are as follows: phase a is 0.1% aqueous trifluoroacetic acid and phase B is 100% acetonitrile; the gradient is: 0-30min. The Fe-N-Boc prepared was dissolved in 20mL 5:2 (v/v) dichloromethane: after reacting in trifluoroacetic acid for 2 hours at room temperature, the solvent is removed by rotary evaporation, and then 20mL of dichloromethane is added for rotary drying, and the reaction is repeated for 3 times, thus obtaining pure Fe-N-NH 2 (with the molecular weight of 360.2). 0.2mM Fe-N-NH 2 was mixed with 0.3mM glutaric anhydride, triethylamine was added to make the solution basic (pH=9), 50℃reflux was carried out in 20mL of methylene chloride for 12h, the methylene chloride was dried by spinning after the reaction was completed, and the methylene chloride was washed three times to obtain purer 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 under the condition of 20mL DMF as a solvent for 12 hours to obtain a crude product Fe-NHS ester. And (3) purifying the crude product by semi-prepared liquid phase to obtain pure Fe-NHS ester. The semi-preparation liquid phase conditions are as follows: phase a is 0.1% aqueous trifluoroacetic acid and phase B is 100% acetonitrile; the gradient is: 0-30min,30-60% B.
(3) Enrichment of plasma membrane proteins of Hela cells using Fe-N-NHS esters:
HeLa cell culture medium, 5X10 7, was blotted, washed twice with PBS, and 10mL of 0.25mg/mL Fe-N-NHS ester/PBS solution was added and labeled on a4 degree shaker for 5min. The labeled reagent was blotted, 10mL of 100mM glycine/PBS solution was added, the mixture was incubated in a4℃shaker for 10min to quench the reaction, and after the quenching reagent was blotted, 10mL TBS buffer was added and washed 1 time. RIPA (strong) lysate containing 1% (v/v) protease inhibitor is added at a ratio of 1mL per 2×10 7 cells, and sonicated at 120W power for 2min to extract protein complexes. Total protein concentration was measured using BCA kit. silicon-based-CB [7] is activated three times with 1mL RIPA, added to the protein suspension, enriched for 2h at room temperature, and the supernatant removed by centrifugation. To the material, 1mL of 200mM dithiothreitol was added, and the denaturation reduction of the protein complex sample was performed in a 56-degree water bath for 30 min. The supernatant was removed by centrifugation and the primary material was washed with 1mL of 50mM ammonium bicarbonate solution; 1mL200mM iodoacetamide is added to carry out alkylation reaction for 30min in dark place; the supernatant was removed by centrifugation and washed multiple times with 1mL of 50mM ammonium bicarbonate solution; finally adding 2 mug trypsin (trypsin: protein complex mass ratio=1:25) for enzymolysis for 14h; the supernatant, i.e., the peptide fragment sample, was collected by centrifugation at 16000 g. The peptide samples obtained were lyophilized and reconstituted with 0.1% (v/v) formic acid and data were collected using liquid chromatography-mass spectrometry. All mass spectrum collected data are searched by adopting MaxQuant-1.6.5.0 with built-in Andromeda search engines, an experimental group and a blank control group perform nonstandard quantitative library searching, data processing is performed on library searching results by Perseus-1.5.8.5 software, differential proteins with quantitative intensity ratio more than 2 and number of Unique peptides more than or equal to 3 are found, and information such as names, cell distribution and the like of the proteins are annotated in Uniprot websites and DAVID websites.
Example 3
(1) The operation is as shown in the steps (1) and (2) in the example 1
(2) The difference proteins of the cell surface proteomes of the common cell lines K562, U937, HL-60, KG-1 and Molm-13 in hematopathy are analyzed by using Fe-Sulfo-NHS ester enrichment:
Cell culture media of 5X 10 7 K562, U937, HL-60, KG-1, molm-13 were blotted separately, washed twice with PBS, and labeled on a 4℃shaker for 5min with 10mL of 0.25mg/mLFe-Sulfo-NHS ester/PBS solution, respectively. The labeled reagent was blotted, 10mL of 100mM glycine/PBS solution was added, the mixture was incubated in a 4℃shaker for 10min to quench the reaction, and after the quenching reagent was blotted, the mixture was added and washed 1 time with 10mLTBS buffer. RIPA (strong) lysate containing 1% (v/v) protease inhibitor is added at a ratio of 1mL per 2×10 7 cells, and sonicated at 120W power for 2min to extract protein complexes. Total protein concentration was measured using BCA kit. silicon-based-CB [7] is activated three times with 1mL RIPA, added to the protein suspension, enriched for 2h at room temperature, and the supernatant removed by centrifugation. To the material, 1mL of 200mM dithiothreitol was added, and the denaturation reduction of the protein complex sample was performed in a 56-degree water bath for 30 min. The supernatant was removed by centrifugation and the primary material was washed with 1mL of 50mM ammonium bicarbonate solution; 1mL200mM iodoacetamide is added to carry out alkylation reaction for 30min in dark place; the supernatant was removed by centrifugation and washed multiple times with 1mL of 50mM ammonium bicarbonate solution; finally adding 2 mug trypsin (trypsin: protein complex mass ratio=1:25) for enzymolysis for 14h; the supernatant, i.e., the peptide fragment sample, was collected by centrifugation at 16000 g. The peptide samples obtained were lyophilized and reconstituted with 0.1% (v/v) formic acid and data were collected using liquid chromatography-mass spectrometry. All mass spectrum collected data are searched by adopting MaxQuant-1.6.5.0 with built-in Andromeda search engine, no-calibration quantity searching is carried out among experimental groups, absolute quantification is carried out on protein expression abundance in the experimental groups by iBAQ values, data processing is carried out on the searching results by Perseus-1.5.8.5 software, differential proteins with relative quantitative intensity ratio more than 2 and number of Unique peptides more than or equal to 3 are found out, expression abundance of the proteins in the respective groups are annotated on information such as names, cell distribution and the like of the proteins in Uniprot websites and DAVID websites. Cell surface proteins that are co-highly expressed in common cell lines in hematological disorders and that are differentially expressed between individual cell lines are sought.
Example 4
(1) The operation is as shown in the steps (1) and (2) in the example 1
(2) Enrichment of cell surface proteins of K562-TRE3G-ETV6-MECOM cell model by using Fe-Sulfo-NHS ester, and analysis of expression change of K562 cell surface proteome caused by ETV6-MECOM gene:
Cell culture media induced by DOX (experimental group) and not DOX (control group 1) and by DOX (control group 2) and not DOX (control group 3) were blotted off at 5X 10 7 K562-TRE3G-ETV6-MECOM, washed twice with PBS, and labeled on a 4 degree shaker with 10mL of 0.25 mg/mLFe-sulfoNHS ester/PBS solution for 5min. The labeled reagent was blotted, 10mL of 100mM glycine/PBS solution was added, the mixture was incubated in a 4℃shaker for 10min to quench the reaction, and after the quenching reagent was blotted, the mixture was added and washed 1 time with 10mLTBS buffer. RIPA (strong) lysate containing 1% (v/v) protease inhibitor is added at a ratio of 1mL per 2×10 7 cells, and sonicated at 120W power for 2min to extract protein complexes. Total protein concentration was measured using BCA kit. silicon-based-CB [7] is activated three times with 1mL RIPA, added to the protein suspension, enriched for 2h at room temperature, and the supernatant removed by centrifugation. To the material, 1mL of 200mM dithiothreitol was added, and the denaturation reduction of the protein complex sample was performed in a 56-degree water bath for 30 min. The supernatant was removed by centrifugation and the primary material was washed with 1mL of 50mM ammonium bicarbonate solution; 1mL 200mM iodoacetamide is added to carry out alkylation reaction for 30min in dark place; the supernatant was removed by centrifugation and washed multiple times with 1mL of 50mM ammonium bicarbonate solution; finally adding 2 mug trypsin (trypsin: protein complex mass ratio=1:25) for enzymolysis for 14h; the supernatant, i.e., the peptide fragment sample, was collected by centrifugation at 16000 g. The peptide samples obtained were lyophilized and reconstituted with 0.1% (v/v) formic acid and data were collected using liquid chromatography-mass spectrometry. All mass spectrum collected data are searched by adopting MaxQuant-1.6.5.0 with built-in Andromeda search engines, an experimental group and a control group perform nonstandard quantitative library searching, data processing is performed on library searching results by Perseus-1.5.8.5 software, differential proteins with quantitative intensity ratio more than 2 and number of Unique peptides more than or equal to 3 are found, and information such as names, cell distribution and the like of the proteins are annotated in Uniprot websites and DAVID websites. The differential cell surface proteins resulting from the influence of the fusion gene ETV6-MECOM were sought.
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.
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