CN114437176B - 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 PDFInfo
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Abstract
The invention discloses a novel optical labeling reagent and application thereof in cell surface proteome and N-glycosylation enrichment analysis, and discloses a novel optical labeling reagent C16-MBP for the first time, wherein the C16-MBP 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 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 optical labeling reagent has important scientific significance and commercial value.
Description
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 broad 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 by 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 multiplex enrichment may severely reduce the scale of identification.
Disclosure of Invention
In view of the above, the present invention aims to overcome the above technical problems in the prior art, and it is an object of the present invention to provide a novel optical labeling reagent for the purpose of efficiently labeling and conveniently enriching cell surface proteins and their glycosylation. 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 combination 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 purpose of the invention is realized by the following technical scheme:
the first aspect of the invention provides a novel light labeling reagent C16-MBP.
Further, the structural formula of the light labeling reagent C16-MBP is shown as the formula I:
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 benzophenone group and adjacent membrane protein are subjected to covalent reaction under 365nm wavelength ultraviolet light, and the specific enrichment of the cell membrane surface protein is realized.
In the present invention, the "C16-MBP", together with 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-crosslinking 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 optical labeling reagent C16-MBP according to 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 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 and purifying fractions, and drying 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:
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 11.25mM;
preferably, the amount of 4- (N-maleimido) benzophenone in step (1) is 6.24mg and 22.50mM;
preferably, the PBS solution in the step (1) is used in an amount of 1mL;
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 30min;
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 the novel light labeling reagent C16-MBP and cell membrane protein under 365nm wavelength ultraviolet light illumination, and then the selectivity and the broad-spectrum enrichment of various cell membrane surface proteins are realized through the affinity effect of streptavidin modified magnetic beads and biotin groups in the 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 of 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), and centrifuging after denaturation to take supernatant for SDS-polyacrylamide gel electrophoresis.
Further, the final concentration of the novel light labeling reagent C16-MBP in the step 1) is 10-50 mu 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 10-30min;
more preferably, the incubation in step 1) is at 4 ℃ for 20min;
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 centrifuging and removing the supernatant in the step 3) are 1000g and 3min;
preferably, the dosage of the RIPA lysate 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 5min;
preferably, the centrifugation conditions for centrifuging the supernatant in the step 3) are 16000g and 10min;
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 1h;
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 step 5) is carried out at 95 ℃ for 10min.
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:
(1) 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 invention 4 HCO 3 Cleaning the solution, removing non-specific adsorption on the magnetic beads through a cleaning step, and adding a probe without C16 into a control group;
(2) after cleaning, carrying out magnetic separation, removing supernatant, carrying out trypsin enzymolysis, after magnetic separation, removing magnetic beads, obtaining supernatant and enzymolysis products in the supernatant, and desalting the enzymolysis products through a desalting small column to obtain eluent;
(3) performing mass spectrum quantitative analysis on the eluent obtained in the step (2), comparing the quantitative difference of the proteins identified by the experimental group and the control group, and subtracting the non-specifically adsorbed non-membrane protein from the calorific value to obtain the cell membrane surface related protein 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, the RIPA lysate, the PBS solution and the NH in the step (1) 4 HCO 3 The dosage of the solution is 200 mu L;
preferably, the trypsin enzymolysis in the step (2) 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 10mM;
more preferably, the condition of adding the dithiothreitol water bath is 56 ℃ and 1h;
more preferably, the final concentration of iodoacetamide is 50mM;
more preferably, the standing condition is dark place and 30min;
more preferably, the amount of trypsin is 1 μ g;
more preferably, the conditions of adding the trypsin water bath are 37 ℃ and 12h;
preferably, the desalting column is a C18 Zip-Tips desalting column.
The method provided by the invention has 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 365nm wavelength ultraviolet light for crosslinking, and then uses streptavidin modified 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 reagents, 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 20min; the 365nm wavelength ultraviolet crosslinking power is 150W, the irradiation time is 1-3min, and the preferable time is 1min; after the incubation is finished, collecting magnetic beads through magnetic separation and removing supernatant, wherein the magnetic beads are sequentially washed by 200 mu L of RIPA (strong) solution and 200 mu L of PBS solution for three times 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; one biotin group can be specifically combined with the commercialized streptavidin modified magnetic beads, and finally, the specific enrichment of cell surface membrane proteins is realized.
(2) Compared with other enrichment methods, the novel cell surface membrane protein enrichment identification method based on the C16-MBP chemical marker provided by the invention has the following three advantages:
(1) compared with a classical enrichment method utilizing ultracentrifugation, the method based on the C16-MBP reagent marking can quickly mark and enrich the cell surface membrane protein, save a large amount of time and initial cell amount, shorten the experimental time and simultaneously mark and enrich the cell membrane surface protein unbiased, and has important significance for enrichment identification of the cell surface membrane protein and discovery of drug targets;
(2) compared with hydrazide chemistry, click chemistry and an enzyme labeling method based on cell surface glycan, the method based on the labeling of the C16-MBP reagent 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 multiple enrichment process 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 enrichment process to realize separation and analysis of glycopeptide, in addition, the efficiency of C16-MBP reagent labeling is higher, and the selectivity and sensitivity are better;
(3) the heptapeptide chain unit is used as a connecting arm for connecting a cell membrane targeting group, a photocrosslinking group and biotin in a C16-MBP reagent, so that the solubility of the probe can be effectively increased, and the probe is easier to target a cell membrane due to the positively charged property of the heptapeptide chain unit, so that 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 cell surface membrane protein enrichment method of 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: 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: volcano maps of cell surface membrane proteins in HT22 cells identified by the method of the 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 which meets the calorific value standard to obtain the first 10 enrichment items;
FIG. 6 is a graph showing the quantitative reproducibility evaluation of cell surface membrane proteins obtained from four technical repeats of the method of the present invention, wherein the numbers in the lower left box represent Pearson correlation coefficients 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 cell surface membrane glycoprotein GO Biological Process and GO Molecular Function analysis obtained by the method of the invention by four technical repeat groups obtain the first 10 enrichment items, and the B picture is as follows: 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 a novel photo-labelling reagent C16-MBP
The structural formula of the new photo-labeling reagent C16-MBP prepared in the embodiment is shown in formula I.
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 synthesis steps of the compound 2 (C16-CRRRRCK-PEG 6-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 30min;
(3) Amino acid linkage: weighing the calculated amount of C-terminal first amino acid Fmoc-Asp (otBu) -NH 2 Adding 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: solution A (6% ninhydrin in ethanol), solution B (80% phenol in ethanol), solution C (2% 0.001M KCN in pyridine), adding small amount of the reacted resin, adding 2-3 drops of solution A, solution B, and solution C, respectively, heating at 105-110 deg.C for 5min, if the solution has blue color, or the resin has blue color and red brown color, indicating that there is free amino group, otherwise indicating complete connection;
(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 carrying out suction filtration on the reaction solution to obtain a trifluoroacetic acid solution of the 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) mass analyze peptide sequence, molecular weight and chemical purity of target compound.
(14) The structural formula of the finally synthesized compound 2 (C16-CRRRRCK-PEG 6-biotin) is shown as a formula II:
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 (20 w), wherein the specific operation steps are as follows:
the 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) and mixed uniformly, and the above mixed sample was subjected to ultrasonic treatment in an ice-water bath. After 30 minutes of pulsed sonication (1 s on, 1s off), the crude product was purified using a desalting column. Collecting the purified fraction, and freeze-drying. 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) respectively and mixed uniformly, and the above mixed sample was subjected to ultrasonic treatment in an ice-water bath. After 30 minutes of pulsed sonication (1 s on, 1s off), the crude product was purified using a desalting column as a control in the subsequent examples. And (3) determining the molecular weight of the C16-PEP-biotin and MBP-biotin probes 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 220nm.
The specific reaction formula of the novel light labeling reagent C16-MBP is as follows:
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 prepared cell membrane localization probe (new light labeling reagent C16-MBP) 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 15cm cell culture dishes were grown to approximately 80% plating rate, the medium was removed from the dishes and the cells were washed 3 times (5 mL each) with ice PBS (4 ℃ C.) to remove excess medium. C16-MBP reagent (final concentration of 20. Mu.M) was added, 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 1min. 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 approximately 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 1h. 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 had significantly fewer 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 without C16 under the same experimental conditions had significantly reduced probe protein bands compared to the experimental group. This is because the control group of probes 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 the cell membrane surface protein.
Example 3 proteomic mass spectrometry analysis of cell membrane surface proteins in C16-MBP reagent-enriched cells
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 NH 4 HCO 3 Each 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 into a 56 ℃ water bath for reduction for 1 hour, then adding 50mM (final concentration) iodoacetamide, placing the mixture in the dark for alkylation treatment for 30min, adding 1 mu g of trypsin into the denatured protein, placing the mixture in a 37 ℃ water bath for incubation for 12 hours, and obtaining supernatant and enzymolysis products in the supernatant after magnetic separation and magnetic bead removal. Desalting the peptide segment of the enzymolysis product by 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, respectively 4 HCO 3 Each 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 20cm long C18 reverse phase chromatography packing packed column (packing diameter 1.9 μm, column inner diameter 75 μm) and the separation of the sample is achieved at a flow rate of 300 nL/min. The scanning range of the primary mass spectrometry is set as350-1500m/z, and the resolution is 60000. The spray voltage was 2.2kV and the ion transfer tube temperature was 320 ℃. AGC is 300% (3 is multiplied by 10) 6 ) The maximum injection time is 50ms and the dynamic exclusion time is 45s. For MS2, resolution was set to 15000, AGC to 75% (7.5 e) 4 ) The maximum injection time is 22ms. The first 10 high parent ions (charge 2-6) were selected for mass spectrometry. The dynamic exclusion time was set to 30s. Allowable mass deviation of. + -.10 ppm and parent ion intensity threshold of 2X 10 4 . For parent ion fragmentation in HCD mode, 30% of the collision energy was used. For FAIMS experiments, the 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 4 C16-MBP reagent enriched cell Membrane surface glycosylation proteome Mass Spectrometry
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 is digested and directly subjected to MS analysis, and the complete N-glycopeptide is separated from the protein due to the fact that the protein is large in molecular weight and is not inhibited by non-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 (38)
1. A light labeling reagent C16-MBP, wherein the structural formula of the light labeling reagent C16-MBP is shown as the formula I:
formula I.
2. A method for preparing the photo-labeling reagent C16-MBP according to claim 1, characterized in that it 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 light labeling reagent C16-MBP in the claim 1;
the structural formula of the C16-CRRRRCK-PEG6-biotin is shown as the formula II:
and (5) formula II.
3. The method of claim 2, wherein the amount of C16-CRRRRCK-PEG6-biotin used in step (1) is 20 mg.
4. The process of claim 2, wherein the amount of 4- (N-maleimido) benzophenone used in step (1) is 6.24 mg.
5. The method of claim 2, wherein the amount of the PBS solution used in step (1) is 1 mL.
6. The method of claim 2, wherein the sonication in step (2) is pulsed ultrasound, 1s on, and 1s off.
7. The method of claim 2, wherein the time of the ultrasonic treatment in step (2) is 30 min.
8. The method according to claim 2, wherein the drying in step (3) is by freeze drying.
9. Use of the light labeling reagent C16-MBP of claim 1 in cell membrane surface protein and glycosylation enrichment assays thereof.
10. The use of claim 9, wherein the covalent coupling between the C16-MBP and the cell membrane proteins occurs under uv illumination with a wavelength of 365nm, and the selective and broad-spectrum enrichment of various cell membrane surface proteins is achieved by the affinity of streptavidin-modified magnetic beads with biotin groups in the C16-MBP.
11. A cell membrane surface protein and glycosylation enrichment analysis method thereof based on the light labeling reagent C16-MBP of claim 1, characterized in that the method comprises the following steps:
1) After washing a cell sample to be tested by using a PBS solution, adding the 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.
12. The method according to claim 11, wherein the final concentration of the photo-labeling reagent C16-MBP in step 1) is 10-50 μ M.
13. The method according to claim 12, wherein the final concentration of the photo-labeling reagent C16-MBP in step 1) is 20 μ M.
14. The method according to claim 11, wherein the incubation in step 1) is performed at 4 ℃ for 10-30 min.
15. The method according to claim 14, wherein the incubation in step 1) is performed at 4 ℃ for 20 min.
16. The method as claimed in claim 11, wherein the conditions of the ultraviolet light crosslinking in step 2) are 365nm, 150W and 1-5min of irradiation time.
17. The method as claimed in claim 16, wherein the conditions of the uv crosslinking in step 2) are uv wavelength 365nm, power 150W, irradiation time 1min.
18. The method of claim 11, wherein the centrifugation conditions for centrifugation to remove supernatant in step 3) are 1000g, 3 min.
19. The method of claim 11, wherein the RIPA lysate is administered in an amount of 350 to 450 μ L in step 3).
20. The method of claim 19, wherein the RIPA lysate is used in step 3) in an amount of 400 μ L.
21. The method of claim 11, wherein the sonication in step 3) is pulsed ultrasound, 2s on, and 2s off.
22. The method of claim 11, wherein the sonication in step 3) is performed for 5 min.
23. The method according to claim 11, wherein the centrifugation conditions for centrifugation of the supernatant in step 3) are 16000g, 10min.
24. The method of claim 11, wherein the streptavidin-modified magnetic beads in step 4) are present in an amount of 50 μ L.
25. The method of claim 11, wherein the incubation in step 4) is at 4 ℃ and 1h.
26. The method of claim 11, wherein the amount of β -mercaptoethanol used in step 5) is 2 μ L.
27. The method of claim 11, wherein the loading buffer in step 5) is used in an amount of 10 μ L.
28. The method according to claim 11, wherein the denaturation in step 5) is carried out at 95 ℃ for 10min.
29. A mass spectrometric identification method of cell membrane surface proteins and their glycosylation enrichment based on the photo-labeling reagent C16-MBP of claim 1, characterized in that it comprises the following steps:
(1) 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 claim 11 4 HCO 3 Cleaning the solution, removing non-specific adsorption on the magnetic beads through a cleaning step, and adding a probe without C16 into a control group;
(2) after cleaning, carrying out magnetic separation, removing supernatant, carrying out trypsin enzymolysis, after magnetic separation, removing magnetic beads, obtaining supernatant and enzymolysis products in the supernatant, and desalting the enzymolysis products through a desalting small column to obtain eluent;
(3) performing mass spectrum quantitative analysis on the eluent obtained in the step (2), comparing the quantitative difference of the proteins identified by the experimental group and the control group, and subtracting the non-specifically adsorbed non-membrane protein from the calorific value to obtain the cell membrane surface related protein 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.
30. The method of claim 29, wherein the RIPA lysate, PBS solution, NH of step (1) 4 HCO 3 The amount of the solution was 200. Mu.L each.
31. The method of claim 29, wherein the trypsinization in step (2) comprises the steps of: adding the mixture into a dithiothreitol water bath for reduction, adding iodoacetamide after reduction, standing for alkylation treatment to obtain denatured protein, and adding the denatured protein into a trypsin water bath for incubation.
32. The method of claim 31, wherein the dithiothreitol is present at a final concentration of 10 mM.
33. The method of claim 31, wherein the aqueous bath of dithiothreitol is added at 56 ℃ and 1h.
34. The method of claim 31, wherein the iodoacetamide is present at a final concentration of 50 mM.
35. The method of claim 31, wherein the standing condition is dark for 30 min.
36. The method of claim 31, wherein the amount of trypsin is 1 μ g.
37. The method of claim 31, wherein the conditions for adding the trypsin water bath are 37 ℃ and 12 h.
38. The method of claim 29, wherein the desalination cartridge of step (2) is a C18 Zip-Tips desalination cartridge.
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