CN114437176A - Novel light labeling reagent and application thereof in cell surface proteome and N-glycosylation enrichment analysis - Google Patents

Novel light labeling reagent and application thereof in cell surface proteome and N-glycosylation enrichment analysis Download PDF

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CN114437176A
CN114437176A CN202210110459.1A CN202210110459A CN114437176A CN 114437176 A CN114437176 A CN 114437176A CN 202210110459 A CN202210110459 A CN 202210110459A CN 114437176 A CN114437176 A CN 114437176A
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张万军
秦伟捷
李圆圆
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

The invention discloses a novel light labeling reagent and application thereof in cell surface proteome and N-glycosylation enrichment analysis, and discloses a novel light labeling reagent C16-MBP for the first time, wherein the C16-MBP reagent mainly comprises four parts, namely a membrane targeting group, a heptapeptide chain group, a biotin group and a light crosslinking group, and the C16-MBP reagent can be applied to enrichment identification of cell membrane surface protein and glycosylation thereof, so that the purposes of high-efficiency labeling and convenient enrichment of cell surface protein and glycosylation thereof are realized, and the light labeling reagent has important scientific significance and commercial value.

Description

Novel light labeling reagent and application thereof in cell surface proteome and N-glycosylation enrichment analysis
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a novel light labeling reagent and application thereof in cell surface proteome and N-glycosylation enrichment analysis, wherein the novel light labeling reagent is C16-MBP.
Background
The cell surface proteome is a class of proteins encoded by 25% of the protein-encoding genes in an organism, and plays a key role in regulating the communication of cells with the surrounding environment. Many membrane proteins are glycosylated and play key roles in many cellular functions and activities, such as cell-cell interactions, pathogen recognition, ion transport and signal transduction. These proteins include a number of cell surface receptors, ion channels and transporters, which account for approximately 70% of all FDA-approved drug targets and thus may reflect the pharmacological relevance of cell surface proteins. Furthermore, glycosylation of cell surface proteins is critical for cell adhesion, migration, and regulation of immune responses. Aberrant protein glycosylation is considered to be a hallmark of cancer and an important target for immunotherapy. Therefore, a thorough analysis of cell surface proteins and their glycosylation may help to better understand their function in various cellular activities and disease progression, thereby helping to discover new biomarkers and drug targets. However, the natural expression levels of most membrane proteins are low compared to intracellular proteins. The difficulty of its highly specific purification has further hampered the understanding of its structure and function.
In order to efficiently isolate cell surface proteins, methods widely reported in recent years can be roughly classified into two major categories, one relying on the physicochemical properties of cell surface proteins and the other relying on the labeling of cell surface proteins. In contrast to high speed centrifugation methods that rely on physical-chemical property separation and detergents based on assisted lysis, enzymatic labeling methods that employ hydrazide chemistry, "click chemistry" and cell surface polysaccharides, although successful in identifying hundreds of cell surface proteins, have still unsatisfactory proteome coverage and selectivity due to the limited cell surface targeting capabilities of existing methods. Furthermore, chemical/enzymatic labeling may alter the composition and structure of glycans, making it difficult to fully elucidate glycans/glycopeptides for mass spectrometry (MS analysis). Although the above limitations can be partially addressed by tandem enrichment of cell surface proteins and their intact glycopeptides, loss of sample during multiple enrichment can severely reduce the scale of identification.
Disclosure of Invention
In view of the above, in order to overcome the above technical problems existing in the prior art, the present invention aims to provide a novel photo-labeling reagent for the purpose of efficiently labeling and conveniently enriching cell surface proteins and glycosylation thereof. The large-scale enrichment of the ultraviolet cross-linked cell membrane surface protein and glycosylation thereof by the C16-MBP reagent is realized through the membrane targeting function of palmitic acid and the covalent binding of a 4- (N-Maleimide) Benzophenone (MBP) group and a probe adjacent protein on a cell membrane, and the C16-MBP reagent mainly comprises four parts, namely a membrane targeting group, a heptapeptide chain group, a biotin group and a photocrosslinking group.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect of the invention, a novel photo-labelling reagent, C16-MBP, is provided.
Further, the structural formula of the light labeling reagent C16-MBP is shown as formula I:
Figure BDA0003494933670000021
furthermore, a part of the novel light labeling reagent C16-MBP is palmitic acid, has a cell membrane targeting function, and a part of the novel light labeling reagent C16-MBP comprises a biotin group and can be specifically combined with a commercialized streptavidin modified magnetic bead, and the novel light labeling reagent C16-MBP also comprises a benzophenone group, so that the novel light labeling reagent and adjacent membrane protein can generate covalent reaction under 365nm wavelength ultraviolet light, and the specific enrichment of the cell membrane surface protein can be realized.
In the present invention, the "C16-MBP", which is the same as the "novel photo labeling reagent", "novel photo labeling reagent C16-MBP", "C16-MBP reagent", "photo labeling reagent C16-MBP", "photo labeling reagent", "cell membrane localization probe C16-MBP" and "compound 1", refers to a novel photo labeling reagent synthesized in the examples of the present invention, which contains a photo cross-linking group, a biotin group and a palmitic acid membrane targeting group, and can be used in the identification of cell surface proteome and N-glycosylation enrichment.
In a second aspect, the present invention provides a method for preparing the novel photo-labeling reagent C16-MBP of the first aspect of the present invention.
Further, the method comprises the steps of:
(1) respectively dissolving C16-CRRRRCK-PEG6-biotin and 4- (N-maleimide) benzophenone in PBS solution and mixing to obtain mixed solution;
(2) carrying out ultrasonic treatment on the mixed solution in the step (1) in an ice-water bath to obtain a crude product;
(3) purifying the crude product in the step (2) by using a desalting column, collecting and drying the purified fraction to obtain a novel light labeling reagent C16-MBP in the first aspect of the invention;
preferably, the structural formula of the C16-CRRRRCK-PEG6-biotin is shown as the formula II:
Figure BDA0003494933670000031
in the invention, the compound 2 is C16-CRRRRCK-PEG6-biotin, which is called C16-PEP-biotin for short and is obtained by self-design and synthesis; the compound 3 is 4- (N-maleimido) benzophenone from Sigma-Aldrich, CAS #: 92944-71-3.
Further, the dosage of the C16-CRRRRCK-PEG6-biotin in the step (1) is 20mg and 11.25 mM;
preferably, the amount of 4- (N-maleimido) benzophenone in step (1) is 6.24mg and 22.50 mM;
preferably, the PBS solution in the step (1) is used in an amount of 1 mL;
preferably, the ultrasonic treatment in the step (2) is pulsed ultrasonic, 1s on and 1s off;
preferably, the time of the ultrasonic treatment in the step (2) is 30 min;
preferably, the drying in step (3) is freeze drying.
The third aspect of the invention provides the application of the novel light labeling reagent C16-MBP in the cell membrane surface protein and glycosylation enrichment analysis thereof.
Furthermore, covalent coupling is generated between a novel light labeling reagent C16-MBP and cell membrane protein under 365nm wavelength ultraviolet light illumination, and then selective and broad-spectrum enrichment of various cell membrane surface proteins is realized through the affinity effect of streptavidin modified magnetic beads and biotin groups in C16-MBP.
The fourth aspect of the invention provides the use of the novel light labelling reagent C16-MBP according to the first aspect of the invention in the mass spectrometric identification of cell membrane surface proteins and their glycosylation enrichment.
The fifth aspect of the present invention provides a cell membrane surface protein based on the novel light labeling reagent C16-MBP of the first aspect of the present invention and a glycosylation enrichment analysis method thereof.
Further, the method comprises the steps of:
1) after a cell sample to be detected is washed by PBS solution, adding a novel light labeling reagent C16-MBP in the first aspect of the invention for incubation;
2) discarding the solution after the incubation is finished, and carrying out ultraviolet crosslinking after the PBS solution is cleaned;
3) after ultraviolet light crosslinking, collecting cells, centrifuging to remove supernatant, adding RIPA lysate to lyse the cells, performing ultrasonic treatment, and centrifuging to obtain supernatant;
4) adding streptavidin modified magnetic beads into the supernatant obtained in the step 3) for incubation, performing magnetic separation after incubation, removing the supernatant, respectively cleaning RIPA lysate and PBS solution, and performing magnetic separation to remove the supernatant to obtain magnetic beads for capturing purified cell membrane surface proteins;
5) adding beta-mercaptoethanol and a loading buffer solution into the magnetic beads obtained in the step 4), denaturing, centrifuging, and taking a supernatant to perform SDS-polyacrylamide gel electrophoresis.
Further, the final concentration of the novel light labeling reagent C16-MBP in the step 1) is 10-50 μ M;
preferably, the final concentration of the novel photo-labelling reagent C16-MBP in step 1) is 20. mu.M;
preferably, the incubation condition in step 1) is 4 ℃ for 10-30 min;
more preferably, the incubation in step 1) is at 4 ℃ for 20 min;
preferably, the ultraviolet light crosslinking conditions in the step 2) are 365nm of ultraviolet light wavelength, 150W of power and 1-5min of irradiation time;
more preferably, the ultraviolet light crosslinking conditions in the step 2) are 365nm of ultraviolet light wavelength, 150W of power and 1min of irradiation time;
preferably, the centrifugation conditions for centrifuging and removing the supernatant in the step 3) are 1000g and 3 min;
preferably, the dosage of the RIPA lysis solution in the step 3) is 350-450 mu L;
more preferably, the dosage of the RIPA lysate in the step 3) is 400 mu L;
preferably, the ultrasonic treatment mode in the step 3) is pulse ultrasonic, 2s is on, and 2s is off;
preferably, the time of the ultrasonic treatment in the step 3) is 5 min;
preferably, the centrifugation conditions for centrifuging the supernatant in the step 3) are 16000g and 10 min;
preferably, the dosage of the streptavidin-immobilized magnetic beads in the step 4) is 50 μ L;
preferably, the incubation in step 4) is carried out at 4 ℃ for 1 h;
preferably, the dosage of the beta-mercaptoethanol in the step 5) is 2 mu L;
preferably, the dosage of the loading buffer solution in the step 5) is 10 mu L;
preferably, the denaturation in step 5) is carried out at 95 ℃ for 10 min.
The sixth aspect of the invention provides a mass spectrometry identification method based on the cell membrane surface protein of the novel light labeling reagent C16-MBP and glycosylation enrichment thereof.
Further, the method comprises the steps of:
the experimental group is that RIPA lysis solution, PBS solution and NH are sequentially added into the magnetic beads obtained in the step 4) of the method of the fifth aspect of the invention4HCO3Washing the solution, removing non-specific adsorption on the magnetic beads through a washing step, wherein a control group is added with a probe without C16;
magnetic separation is carried out after cleaning, supernatant is discarded, trypsin enzymolysis is carried out, magnetic beads are discarded after magnetic separation, supernatant and enzymolysis products in the supernatant are obtained, and the enzymolysis products are desalted through a small desalting column to obtain eluent;
carrying out mass spectrum quantitative analysis on the eluent obtained in the step two, comparing the quantitative difference of the identified proteins of the experimental group and the control group, and deducting non-specifically adsorbed non-membrane proteins from the calorific value to obtain the cell membrane surface related proteins with high confidence; directly identifying N-glycosylated protein in trypsin digested peptide without enriching N-glycopeptide by adopting a high-field asymmetric waveform ion mobility spectrometry technology.
Further, in the step (i), the RIPA lysate, the PBS solution and the NH4HCO3The dosage of the solution is 200 mu L;
preferably, the trypsin enzymolysis in step (II) comprises the following steps: adding dithiothreitol water bath for reduction, adding iodoacetamide after reduction, standing for alkylation treatment to obtain denatured protein, and adding trypsin into the denatured protein for incubation;
more preferably, the final concentration of dithiothreitol is 10 mM;
more preferably, the condition of adding the dithiothreitol water bath is 56 ℃ and 1 h;
more preferably, the final concentration of iodoacetamide is 50 mM;
more preferably, the standing condition is dark place and 30 min;
more preferably, the amount of trypsin is 1 μ g;
more preferably, the conditions for adding the trypsin water bath are 37 ℃ and 12 hours;
preferably, the desalting column is a C18 Zip-Tips desalting column.
The method provided by the invention comprises the following flows and principles: after a cell to be detected in a culture dish is added with a C16-MBP reagent for incubation, ultraviolet irradiation with 365nm wavelength is used for promoting a benzophenone group in the C16-MBP reagent to form a covalent bond with adjacent protein, so that biotinylation of the cell surface is realized. Then collecting cells, carrying out ultrasonic lysis on the cells to obtain a sample, incubating the sample with streptavidin modified magnetic beads, and cleaning to remove non-specifically adsorbed proteins and other molecules, thereby realizing the specific enrichment of cell surface membrane proteins. And subsequently, by combining a high-field asymmetric waveform ion mobility spectrometry (FAIMS) technology, the corresponding glycosylated protein is directly analyzed from the enriched membrane protein under the condition of not carrying out tandem N-glycopeptide enrichment.
Further, in order to obtain a more accurate and reliable cell surface membrane protein identification result, the invention adopts a probe without C16, namely without a membrane targeting function, to incubate with cells, then adopts the same experimental conditions for a control group sample, uses ultraviolet light with the wavelength of 365nm to crosslink, and then uses streptavidin to modify magnetic beads to enrich cell surface membrane protein. However, unlike the experimental group, since the control group had no membrane targeting function, 0.05% Tween 20 was added in the washing step after the incubation of the reagent, thereby removing the probe from the cells. The resulting product in the subsequent enrichment will therefore no longer contain cell surface membrane proteins, but only proteins that are non-specifically adsorbed to the magnetic beads. On the basis, samples of the experimental group and the control group are subjected to mass spectrometry after being subjected to enzymolysis into peptide fragments by using trypsin. Therefore, more accurate cell surface membrane protein identification results can be obtained by identifying the difference of the types and the contents of the proteins in the experimental group and the control group, and if the protein in the control group is not identified or the content of the protein is obviously lower than that of the protein in the experimental group, the cell surface membrane protein can be considered to be high-credibility cell surface membrane protein.
Further, in the above method, the incubation time of the C16-MBP reagent is 10-30min, preferably 20 min; the 365nm wavelength ultraviolet crosslinking power is 150W, the irradiation time is 1-3min, and the preferable time is 1 min; after the incubation is finished, magnetic beads can be collected by magnetic separation and supernatant is discarded, and the magnetic beads are sequentially washed three times by 200 mu L of RIPA (strong) solution and 200 mu L of PBS solution respectively; the supernatant was discarded by magnetic separation to obtain magnetic beads in which cell surface membrane proteins were captured. Subsequently, 2. mu.L of 0.5. mu.g/. mu.L of tryptin was added for proteome analysis.
Compared with the prior art, the invention has the advantages and beneficial effects that:
(1) the invention not only provides a new light labeling reagent C16-MBP for the field, but also provides a new enrichment identification method of cell membrane surface protein and glycosylation thereof based on the chemical labeling of the light labeling reagent C16-MBP, wherein the C16-MBP reagent adopted in the method contains a C16 group and can specifically target cell membranes; a polyethylene glycol (PEG) spacer for increasing the water solubility of the agent; two 4- (N-Maleimido) Benzophenone (MBP) groups for uv crosslinking with cell surface proteins; a biotin group can be specifically combined with commercial streptavidin modified magnetic beads, and finally specific enrichment of cell surface membrane protein is realized.
(2) Compared with other enrichment methods, the novel cell surface membrane protein enrichment identification method based on the C16-MBP chemical marker has the following three advantages:
compared with the classical enrichment method utilizing ultracentrifugation, the C16-MBP reagent labeling-based method can rapidly label and enrich cell surface membrane proteins, saves a large amount of time and initial cell amount, shortens the experimental time, and simultaneously labels and enriches cell membrane surface proteins unbiased, and has important significance for enrichment identification of the cell surface membrane proteins and discovery of drug targets;
compared with hydrazide chemistry, click chemistry and an enzyme labeling method based on cell surface glycan, the method based on C16-MBP reagent labeling can identify more cell surface proteins, has better cell surface targeting capability and has ideal proteome coverage rate and selectivity. In addition, the chemical/enzymatic labeling of glycan may change its composition and structure, thus leading to MS difficult to fully elucidate glycan/glycopeptide, the significant loss of sample in the process of multiple enrichment may also seriously reduce the scale of identification, and the FAIMS technology can be used to directly analyze the enriched membrane protein, thus avoiding the loss in the process of enrichment to realize separation and analysis of glycopeptide, in addition, the efficiency of labeling C16-MBP reagent is higher, and the selectivity and sensitivity are better;
and thirdly, the heptapeptide chain unit is used as a connecting arm for connecting a cell membrane targeting group, a photocrosslinking group and biotin in the C16-MBP reagent, so that the solubility of the probe can be effectively increased, and meanwhile, the probe is easier to target the cell membrane due to the positively charged property of the heptapeptide chain unit, and the enrichment efficiency of streptomycin modified magnetic beads on cell surface protein is improved.
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FIG. 1 is a flow chart of the experiment of the method for enriching the cell surface membrane protein according to the present invention;
FIG. 2 is a MALDI-TOF-MS characterization chart of the cell membrane localization probe C16-MBP synthesized by the present invention;
FIG. 3 is a fluorescent diagram showing the membrane localization effect of the cell membrane localization probe C16-MBP synthesized in the present invention;
FIG. 4 is a SDS-polyacrylamide gel electrophoresis of the cell surface membrane protein enrichment product of cells according to the method of the present invention; wherein, A is as follows: the control group is obtained by adding no C16-MBP probe, performing no 365nm ultraviolet irradiation or performing no streptavidin modified magnetic bead enrichment treatment in the experimental process, and a B picture is as follows: the control group does not contain the C16 probe and is incubated with cells, so that the effectiveness of the C16-MBP probe on the enrichment of cell surface membrane proteins is verified;
FIG. 5 is a graph showing the results of the assay according to the present invention, wherein A is a graph: volcano maps of cell surface membrane proteins in HT22 cells identified by the method of the present invention (quantitative difference maps of proteins in experimental group and control group); and B, drawing: analyzing the GO cellular component, GO Biological Process and GO Molecular Function of the identified cell surface membrane protein meeting the caloric value standard to obtain the first 10 enrichment items;
FIG. 6 is a graph showing the quantitative reproducibility of the cell surface membrane proteins obtained from four technical repeats of the method of the present invention, wherein the numbers in the lower left box indicate the Pearson correlation coefficient between the two corresponding technical repeats;
FIG. 7 is a graph showing the results of identifying four technical repeats of the method of the present invention, wherein A is a graph: the qualitative reproducibility evaluation of the cell surface membrane protein obtained by the four technical repeats of the method of the invention is shown in figure B: the number of total proteins and corresponding cell surface membrane proteins identified per technical replicate group;
FIG. 8 is a graph showing the results of identifying four technical repeats of the method of the present invention, wherein A is a graph: the method of the invention adopts four technical repeated groups to obtain cell surface membrane glycoprotein GO Biological Process and GO Molecular Function analysis, the first 10 enrichment items are obtained, B picture: the method disclosed by the invention is used for identifying the number of cell surface membrane glycoproteins and glycosylated peptide sections obtained by four technical repeat groups.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of the novel photo-labelling reagent C16-MBP
The structural formula of the new photo-labeling reagent C16-MBP prepared in this example is shown in formula I.
Figure BDA0003494933670000091
1. Firstly, compound 2 for preparing a new light labeling reagent C16-MBP, namely a membrane targeting control probe C16-CRRRRCK-PEG6-biotin (C16-PEP-biotin for short) is synthesized
The specific synthetic steps of the compound 2(C16-CRRRRCK-PEG6-biotin) are as follows:
(1) the peptide chains are linked from the amino acid at the C terminal to the N terminal;
(2) resin activation: taking 2-Cl resin, adding a proper amount of DMF (dimethyl formamide) into a clean and dry reaction tube, and activating for about 30 min;
(3) amino acid linkage: weighing the calculated amount of C-terminal first amino acid Fmoc-Asp (otBu) -NH2Adding 0.5mL of protected DIEA into a reaction tube, adding excessive DMF as a solvent for reaction, and adding different catalysts according to different amino acids;
(4) eluting Fmoc protection;
(5) and (3) detection: in the solid-phase polypeptide synthesis, the connection efficiency is mainly judged by detecting free amino groups on resin, the detection method is a Kaiser method, and the detection result shows blue or reddish brown (Pro, Ser, His) if the free amino groups exist, wherein the Kaiser reagent comprises: taking a small amount of reacted resin from the solution A (6% ninhydrin ethanol solution), the solution B (80% phenol ethanol solution) and the solution C (2% 0.001M KCN pyridine solution), adding 2-3 drops of the solution A, the solution B and the solution C respectively, and heating at the temperature of 105-;
(6) and after the detection is successful, the second amino acid at the C terminal is continuously linked. The method is the same as that of the step (3);
(7) after the amino acid is grafted, adding a proper amount of DMF (dimethyl formamide), adding DIEA (dimethyl EA), adding FITC (fluorescein isothiocyanate) for reaction for 4 hours, and keeping out of the sun;
(8) cutting: cutting with trifluoroacetic acid cutting fluid for 2.5-5h, and filtering the reaction solution to obtain trifluoroacetic acid solution of polypeptide;
(9) and (3) precipitation: precipitating with excessive diethyl ether, centrifuging, eluting the centrifuged sample with diethyl ether for multiple times, and centrifuging to obtain a primary peptide sample;
(10) and (3) purification: purifying the crude peptide by HPLC to obtain high purity;
(11) mass spectrometry (detection);
(12) freeze-drying: liquid nitrogen is rapidly cooled and then lyophilized for use.
(13) Mass analysis (COA): high Performance Liquid Chromatography (HPLC) and Mass Spectrometer (MS) are used for mass analysis of peptide sequence, molecular weight and chemical purity of target compounds.
(14) The structural formula of the finally synthesized compound 2(C16-CRRRRCK-PEG6-biotin) is shown as a formula II:
Figure BDA0003494933670000111
2. synthesis of novel light labeling reagent C16-MBP
The new light labeling reagent C16-MBP is obtained by hydrolysis after Michael addition reaction of compound 2(C16-PEP-biotin) and compound 3(4- (N-maleimide) benzophenone) (from Sigma-Aldrich, CAS #: 92944-71-3) synthesized by the above steps, wherein the feeding molar ratio of the membrane targeting control probe C16-CRRRRCK-PEG6-biotin (C16-PEP-biotin, 20mg, 11.25 μ M) to 4- (N-maleimide) benzophenone (MBP, 6.24mg, 22.50 μ M) is 1: 2, the michael addition reaction is carried out in a water solvent, and the operation steps of the michael addition reaction are as follows: firstly, dissolving a compound 3 in water, then dropwise adding a compound shown as a compound 2, reacting at room temperature overnight, and then performing mechanical ultrasonic treatment for 30min (20w), wherein the specific operation steps are as follows:
C16-CRRRRCK-PEG6-biotin (C16-PEP-biotin, 20mg, 11.25. mu.M) and 4- (N-maleimido) benzophenone (MBP, 6.24mg, 22.50. mu.M) synthesized by the above steps were dissolved in 1mL of PBS (pH 7.4) respectively and mixed well, and the above mixed sample was subjected to ultrasonic treatment in an ice-water bath. After 30 minutes of pulsed sonication (1s on, 1s off), the crude product was purified using a desalting column. The purified fractions were collected and lyophilized. For the non-membrane targeting control probe without C16, CRRRRCK-PEG6-biotin (PEP-biotin, 20mg, 12.99. mu.M) and 4- (N-maleimido) benzophenone (MBP, 7.20mg, 25.98. mu.M) were dissolved in 1mL of PBS (pH 7.4) and mixed well, and the above mixed sample was subjected to ultrasonication in an ice-water bath. After 30 minutes of pulsed sonication (1s on, 1s off), the crude product was purified using a desalting column as a control in the subsequent examples. The molecular weight of the C16-PEP-biotin and MBP-biotin probes is determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The purity of both probes was determined by liquid chromatography using mobile phases A (0.1% trifluoroacetic acid-100% water) and B (0.1% trifluoroacetic acid-100% acetonitrile) at a flow rate of 1 mL/min. The analytical column was SHIMADZU Inertsil ODS-SP, with a size of 4.6X 250mm and a particle size of 5 μm. The detection wavelength was 220 nm.
The specific reaction formula of the novel light labeling reagent C16-MBP is as follows:
Figure BDA0003494933670000121
the experimental flow chart of the cell surface membrane protein enrichment method is shown in figure 1; the MALDI-TOF-MS characterization diagram of the cell membrane positioning probe (novel optical labeling reagent C16-MBP) prepared by the invention is shown in figure 2, and the result shows that the optical labeling reagent C16-MBP containing a membrane targeting group, a heptapeptide chain group, a biotin group and an optical crosslinking group is successfully prepared by the invention; the fluorescence result diagram of the membrane localization effect of the cell membrane localization probe (new light labeling reagent C16-MBP) prepared by the invention is shown in figure 3, and the result shows that the light labeling reagent C16-MBP prepared by the invention can be used for accurate localization of cell surface membrane protein.
Example 2 evaluation of the Effect of C16-MBP reagent on enrichment of cell surface Membrane proteins in cells
When HT22 cells (purchased from Shanghai leaf Biotech, Inc.) in a 15cm cell culture dish were grown to approximately 80% plating rate, the medium was removed from the dish and the cells were washed 3 times (5 mL each) with ice PBS (4 ℃ C.) to remove excess medium. C16-MBP reagent (20 μ M final concentration) was added and incubated at 4 ℃ for 20min, after which the solution was discarded, washed 3 times with PBS, then placed on ice and UV-crosslinked. The wavelength of ultraviolet light is 365nm, the power is 150W, and the irradiation time is 1 min. Cells were scraped into 1.5mL EP tubes with cell scraping, centrifuged at 1000g for 3min to remove supernatant, and then RIPA (Strong) was added to make the total volume about 400. mu.L. Sonicate on ice for about 5min (200W, 2s on, 2s off), centrifuge at 16000g for 10min and aspirate supernatant into a new EP tube. mu.L of streptavidin-immobilized magnetic beads (Thermo) was added and incubated at 4 ℃ for 1 h. The supernatant was discarded by magnetic separation, and the magnetic beads were washed three times with 200. mu.L of RIPA (Strong) and 200. mu.L of PBS, respectively. The supernatant was discarded by magnetic separation, 2. mu.L of beta-mercaptoethanol and 10. mu.L of loading buffer were added, denaturation was carried out at 95 ℃ for 10min, and the supernatant was centrifuged to carry out SDS-polyacrylamide gel electrophoresis.
As shown in FIG. 4A, the control group to which the C16-MBP reagent was not added showed significantly less protein bands compared to the experimental group, demonstrating that the C16-MBP reagent plays a key role in the enrichment of cell surface membrane proteins. In addition, the control histone bands without 365nm ultraviolet light in the experimental process are relatively less, because the 365nm ultraviolet cross-linking probe cannot be covalently combined with cell surface protein, and false positive results cannot be introduced.
As shown in FIG. 4B, the control group probe protein band without C16 was significantly reduced under the same experimental conditions compared to the experimental group. This is because the control group probe without C16 has no membrane targeting function, and can be washed clean by solvent after cell incubation, and the magnetic beads have no captured protein, and only a small amount of non-specific adsorption on the magnetic beads is possible. The experiments prove that the C16-MBP reagent has good selectivity and enrichment effect on cell membrane surface protein.
Example 3 proteomic mass spectrometry analysis of cell membrane surface proteins in cells enriched with C16-MBP reagent
Experimental groups: after obtaining purified cell membrane surface protein by magnetic bead capture using the same experimental conditions as in example 2, the supernatant was discarded by magnetic separation using 200. mu.L of RIPA (Strong), 200. mu.L of PBS solution, and 200. mu.L of 50mM NH4HCO3Each wash was three times. Removing supernatant through magnetic separation, and performing trypsin enzymolysis, wherein the specific operations are as follows: adding 10mM (final concentration) dithiothreitol, reducing in 56 deg.C water bath for 1 hr, adding 50mM (final concentration) iodoacetamide, standing in dark for 30min for alkylation, collecting denatured protein, and adding 1 μ g pancreasAnd (3) putting the protease into a water bath at 37 ℃ for incubation for 12 hours, and obtaining supernatant and an enzymolysis product in the supernatant after magnetic separation and magnetic bead removal. Desalting the peptide segment of the enzymolysis product by using a C18 Zip-Tips desalting small column, and freeze-drying eluent for later use.
Control group: the same amount of cells as in the experimental group was taken and the other operations were identical to those in the experimental group. The supernatant was discarded by magnetic separation, and the magnetic beads were washed with 200. mu.L of RIPA (Strong), 200. mu.L of PBS solution, and 200. mu.L of 50mM NH, respectively4HCO3Each wash was three times.
Mass spectrometry analysis: samples were analyzed using nanoliter liquid chromatography (EASY-nLC 1200) in combination with Orbitrap explooris 480 using a mass spectrometer. Wherein, the liquid chromatography uses a C18 reversed phase chromatography packing column (packing diameter is 1.9 μm, inner diameter of the column is 75 μm) with the length of 20cm, and the separation of the sample is realized at the flow rate of 300 nL/min. The scanning range of the primary mass spectrometry is set to 350-1500m/z, and the resolution is 60000. The spray voltage was 2.2kV and the ion transfer tube temperature was 320 ℃. AGC is 300% (3X 10)6) The maximum injection time is 50ms and the dynamic exclusion time is 45 s. For MS2, resolution was set to 15000 and AGC to 75% (7.5 e)4) The maximum injection time is 22 ms. The first 10 high parent ions (charge 2-6) were selected for mass spectrometry. The dynamic exclusion time was set to 30 s. Allowable mass deviation of. + -.10 ppm and parent ion intensity threshold of 2X 104. For parent ion fragmentation in HCD mode, 30% of the collision energy was used. For FAIMS experiments, analyses were performed using CVs of-45 and-60.
Mass spectrometry data analysis: and (4) performing library searching analysis on the mass spectrum raw data by using MaxQuant software. The protease cleavage pattern was set to "trypsin/P" and allowed to contain a maximum of 2 cleavage sites (missed cleavage sites) per peptide fragment, and a minimum of 6 amino acid residues per peptide fragment. The urethyl modified cysteine (i.e. cysteine blocked by iodoacetamide) was set as a fixed modification, the oxidation of methionine and the N-terminal acetyl modification as variable modifications. For protein identification, the FDR upper limit was set to 1%. Proteins with a P value of less than 0.01 and a fold enrichment of 4 or more were considered to be highly reliable cell membrane surface proteins.
As shown in fig. 5, the method provided by the present invention identified 2835 highly-confident cell membrane surface proteins. GO function analysis is carried out on the proteins, and the result shows that most of the most enriched items are related to the cell membrane surface protein, thereby further proving the reliability and selectivity of the method. The correlation analysis of the cell membrane surface protein obtained by four technical repetitions was further performed, and the results are shown in FIG. 6. The correlation among the technical repetitions can reach more than 0.9, and the experimental repeatability and the reliability of the technology are proved to be good. As shown in fig. 7, 3103 cell surface membrane proteins are identified by the four techniques of the method provided by the invention, 2560 (75.4%) of repeatedly identified cell membrane surface proteins are identified at least twice, and the selectivity of a single experiment is greater than 65%, and the results further prove that the method has high selectivity and broad spectrum in the aspect of large-scale enrichment of cell membrane surface proteins.
Example 4C 16-MBP reagent-enriched Mass Spectrometry of cell Membrane surface glycosylated proteomes
Tandem N-glycopeptide enrichment may be impractical given the limited amount of cell membrane surface protein samples to enrich for. Thus, in this example, the inventors propose to directly identify N-glycosylated proteins in tryptic peptides that are not enriched in N-glycopeptides using high field asymmetric waveform ion mobility spectrometry (FAIMS). The cell surface protein enriched with the C16-MBP probe was digested and directly subjected to MS analysis, and due to its large molecular weight, it was not inhibited by non-glycopeptides, thus allowing the isolation of intact N-glycopeptides.
As shown in fig. 8A, 793 glycoproteins, including 1483 glycopeptides, were identified from the enriched product of the C16-MBP probe by LC-MS analysis, indicating the potential of this strategy in large-scale cell surface N-glycoprotein group analysis. Figure 8B is a molecular functional and biological process analysis of the identified N-glycoprotein, with significant enrichment for molecular functions of "signal receptor activity" (related to signal transduction, receptor activity) and "molecular trafficking activity", as well as "cellular communication", "developmental processes" and "signal transduction", consistent with the reported N-glycoprotein GO function.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (10)

1. A novel light labeling reagent C16-MBP, wherein the structural formula of the light labeling reagent C16-MBP is shown as formula I:
Figure FDA0003494933660000011
2. a method for preparing the novel photo-labelling reagent C16-MBP according to claim 1, which comprises the following steps:
(1) respectively dissolving C16-CRRRRCK-PEG6-biotin and 4- (N-maleimide) benzophenone in a PBS solution and mixing to obtain a mixed solution;
(2) carrying out ultrasonic treatment on the mixed solution in the step (1) in an ice-water bath to obtain a crude product;
(3) purifying the crude product in the step (2) by using a desalting column, collecting the purified fraction, and drying to obtain the novel light labeling reagent C16-MBP of claim 1;
preferably, the structural formula of the C16-CRRRRCK-PEG6-biotin is shown as a formula II:
Figure FDA0003494933660000012
3. the method of claim 2, wherein the amount of C16-CRRRRCK-PEG6-biotin in step (1) is 20mg, 11.25 mM;
preferably, the amount of 4- (N-maleimido) benzophenone in step (1) is 6.24mg and 22.50 mM;
preferably, the PBS solution in the step (1) is used in an amount of 1 mL;
preferably, the ultrasonic treatment in the step (2) is pulsed ultrasonic, 1s on and 1s off;
preferably, the time of the ultrasonic treatment in the step (2) is 30 min;
preferably, the drying in step (3) is freeze drying.
4. The use of the novel light labeling reagent C16-MBP of claim 1 in cell membrane surface protein and glycosylation enrichment assays thereof.
5. The use of claim 4, wherein covalent coupling between the novel light labeling reagent C16-MBP and cell membrane proteins occurs under 365nm ultraviolet light, and selective and broad-spectrum enrichment of various cell membrane surface proteins is achieved through the affinity of streptavidin modified magnetic beads and biotin groups in C16-MBP.
6. Use of the novel photo-labelling reagent C16-MBP according to claim 1 in the mass spectrometric identification of cell membrane surface proteins and their glycosylation enrichment.
7. The cell membrane surface protein and glycosylation enrichment analysis method thereof based on the novel light labeling reagent C16-MBP of claim 1, wherein the method comprises the following steps:
1) after washing a cell sample to be tested by using a PBS solution, adding a novel light labeling reagent C16-MBP of claim 1 for incubation;
2) discarding the solution after the incubation is finished, and performing ultraviolet crosslinking after the PBS solution is cleaned;
3) after ultraviolet light crosslinking, collecting cells, centrifuging to remove supernatant, adding RIPA lysate to lyse the cells, performing ultrasonic treatment, and centrifuging to obtain supernatant;
4) adding streptavidin modified magnetic beads into the supernatant obtained in the step 3) for incubation, performing magnetic separation after incubation, removing the supernatant, respectively cleaning RIPA lysate and PBS solution, and performing magnetic separation to remove the supernatant to obtain magnetic beads for capturing purified cell membrane surface proteins;
5) adding beta-mercaptoethanol and a loading buffer solution into the magnetic beads obtained in the step 4), and centrifuging after denaturation to take supernatant for SDS-polyacrylamide gel electrophoresis.
8. The method according to claim 7, wherein the final concentration of the novel photo-labelling reagent C16-MBP in step 1) is 10-50 μ M;
preferably, the final concentration of the novel photo-labelling reagent C16-MBP in step 1) is 20. mu.M;
preferably, the incubation condition in step 1) is 4 ℃ for 10-30 min;
more preferably, the incubation in step 1) is at 4 ℃ for 20 min;
preferably, the ultraviolet light crosslinking conditions in the step 2) are 365nm of ultraviolet light wavelength, 150W of power and 1-5min of irradiation time;
more preferably, the ultraviolet light crosslinking conditions in the step 2) are 365nm of ultraviolet light, 150W of power and 1min of irradiation time;
preferably, the centrifugation conditions for removing the supernatant by centrifugation in the step 3) are 1000g and 3 min;
preferably, the dosage of the RIPA lysis solution in the step 3) is 350-450 mu L;
more preferably, the dosage of the RIPA lysate in the step 3) is 400 mu L;
preferably, the ultrasonic treatment in the step 3) is pulsed ultrasonic, 2s on and 2s off;
preferably, the time of the ultrasonic treatment in the step 3) is 5 min;
preferably, the centrifugation conditions for centrifuging the supernatant in the step 3) are 16000g and 10 min;
preferably, the dosage of the streptavidin-immobilized magnetic beads in the step 4) is 50 μ L;
preferably, the incubation in step 4) is carried out at 4 ℃ for 1 h;
preferably, the dosage of the beta-mercaptoethanol in the step 5) is 2 mu L;
preferably, the dosage of the loading buffer solution in the step 5) is 10 mu L;
preferably, the denaturation in step 5) is carried out at 95 ℃ for 10 min.
9. A mass spectrometric identification method of cell membrane surface proteins and their glycosylation enrichment based on the novel photo-labeling reagent C16-MBP of claim 1, characterized in that it comprises the following steps:
the experimental group is that RIPA lysis solution, PBS solution and NH are added in sequence into the magnetic beads obtained in the step 4) of the method of claim 84HCO3Washing the solution, removing non-specific adsorption on the magnetic beads through a washing step, wherein a control group is added with a probe without C16;
magnetic separation is carried out after cleaning, supernatant is discarded, trypsin enzymolysis is carried out, magnetic beads are discarded after magnetic separation, supernatant and enzymolysis products in the supernatant are obtained, and the enzymolysis products are desalted through a small desalting column to obtain eluent;
carrying out mass spectrum quantitative analysis on the eluent obtained in the step two, comparing the quantitative difference of the identified proteins of the experimental group and the control group, and deducting non-specifically adsorbed non-membrane proteins from the calorific value to obtain the cell membrane surface related proteins with high confidence; directly identifying N-glycosylated protein in trypsin digested peptide without enriching N-glycopeptide by adopting a high-field asymmetric waveform ion mobility spectrometry technology.
10. The method according to claim 9, wherein the RIPA lysate, the PBS solution, and NH in step (i)4HCO3The dosage of the solution is 200 mu L;
preferably, the trypsin enzymolysis in step (II) comprises the following steps: adding dithiothreitol water bath for reduction, adding iodoacetamide after reduction, standing for alkylation treatment to obtain denatured protein, and adding trypsin into the denatured protein for incubation;
more preferably, the final concentration of dithiothreitol is 10 mM;
more preferably, the condition of adding the dithiothreitol water bath is 56 ℃ and 1 h;
more preferably, the final concentration of iodoacetamide is 50 mM;
more preferably, the standing condition is dark place and 30 min;
more preferably, the amount of trypsin is 1 μ g;
more preferably, the conditions for adding the trypsin water bath are 37 ℃ and 12 hours;
preferably, the desalting column is a C18 Zip-Tips desalting column.
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