CN114577959A - Method for analyzing modification of multiple proteins in biological sample - Google Patents
Method for analyzing modification of multiple proteins in biological sample Download PDFInfo
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- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
Abstract
A method of analyzing multiple protein modifications in a biological sample is disclosed. The method comprises the steps of respectively carrying out peptide enrichment on peptide fragments subjected to protein enzymolysis digestion of a sample to be detected according to the type of protein modification to be detected; mixing the enriched peptide fragments to obtain a protein modification type mixed peptide fragment to be detected; performing one-time DIA collection and/or DDA collection on the mixed peptide fragments; and analyzing the DIA information and/or the DDA information based on a DDA spectrogram library corresponding to the protein modification type peptide fragment to be detected to obtain a protein modification analysis result of the sample to be detected. According to the method, the enriched peptide fragments of the protein modification type to be detected are mixed, and the DDA spectrogram libraries of the peptide fragments are combined, so that the analysis result of multiple protein modifications in a sample to be detected can be obtained through analysis only by acquiring data of the mixed peptide fragments once; the detection period of the modification of various proteins is shortened, the machine time of a mass spectrometer is saved, and the detection cost of the modification of various proteins is reduced.
Description
Technical Field
The application relates to the technical field of protein modification analysis, in particular to a method for analyzing multiple protein modifications in a biological sample.
Background
For a long time, protein modification or the study of protein modification has not attracted sufficient attention, and this situation was not improved until 2004 when the discovery of ubiquitination-mediated protein degradation achieved the Nobel prize. To date, over 200 protein modifications have been found, common protein modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis. Protein modification by adding a modification group to one or more amino acid residues, can change the physical and chemical properties of the protein, thereby influencing the spatial conformation, activity, subcellular localization, protein folding and protein-protein interaction of the protein. Protein modification plays a key role in a variety of cellular processes, such as cell division, proteolysis, signaling, regulatory processes, regulation of gene expression, and protein interactions.
In the field of modified proteome research, it is a common detection technique to perform analysis by using a mass spectrometer after obtaining a modified peptide fragment by enrichment, and the common mass spectrometry scanning modes include a data-dependent acquisition (DDA) mode and a data-independent acquisition (DIA) mode. The DIA combines the characteristics of DDA and Selective Reaction Monitoring (SRM), equally divides the entire scan range into several windows, each window is Selected and fragmented in turn, and collects information of all daughter ions of all parent ions within the window.
In the prior art, each protein modification is detected independently, for example, phosphorylated protein modification and glycosylated protein modification are detected, as shown in fig. 1, if the two protein modifications are required to be detected, phosphorylated enrichment and glycosylated enrichment are required to be performed on polypeptide products obtained by protein extraction and enzymolysis, and phosphorylated peptide fragments and glycosylated peptide fragments are obtained respectively; obtaining a phosphorylation DDA spectrum library by using the phosphorylation peptide segment, and obtaining a glycosylation DDA spectrum library by using the glycosylation peptide segment; then respectively carrying out DIA mode mass spectrum data acquisition on the phosphorylated peptide segments, and analyzing the acquired phosphorylated DIA data based on a phosphorylated DDA spectrogram library to obtain a phosphorylated protein modification detection result of the sample to be detected; and (3) performing DIA-mode mass spectrum data acquisition on the glycosylated peptide section, and analyzing the acquired glycosylated DIA data based on a glycosylated DDA spectrogram library to obtain a glycosylated protein modification detection result of the sample to be detected.
Therefore, once DIA data acquisition is needed for each protein modification detection; if eight of the common protein modifications described above are to be detected, eight DIA data acquisitions are required. However, a single DIA data acquisition requires 2 hours of mass spectrometer machine time to spend, with a small time the cost is about 400 dollars; it is very expensive to detect only eight common protein modifications in a sample, and if more other protein modifications need to be detected, the cost is more expensive, and the detection period is long and the time cost is high.
Therefore, how to analyze or detect multiple protein modifications more efficiently is a problem to be solved in the art.
Disclosure of Invention
It is an object of the present application to provide an improved method for analyzing multiple protein modifications in a biological sample.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the application discloses a method for analyzing multiple protein modifications in a biological sample, which comprises the steps of respectively carrying out corresponding peptide enrichment on peptide fragments subjected to protein enzymolysis digestion according to protein modification types to be detected; the peptide segment of the protein enzymolysis digestion is the peptide segment obtained by carrying out enzymolysis digestion on the protein extracted from a sample to be detected; mixing the enriched peptide fragments to obtain a protein modification type mixed peptide fragment to be detected; performing one-time DIA collection and/or DDA collection on the mixed peptide fragments; and analyzing the acquired DIA information and/or DDA information based on the DDA spectrogram library corresponding to the peptide fragment of the protein modification type to be detected to obtain the protein modification analysis result of the sample to be detected.
The peptide fragments obtained by the enzymatic digestion of the protein are respectively subjected to corresponding peptide fragment enrichment according to the type of the protein modification to be detected, for example, the type of the protein modification to be detected is phosphorylated protein modification, the peptide fragment enrichment is performed according to a phosphorylated peptide fragment enrichment method, the type of the protein modification to be detected is glycosylated protein modification, and the peptide fragment enrichment is performed according to a glycosylated peptide fragment enrichment method.
It should be noted that the key point of the method of the present application is to mix all the enriched peptide fragments together, and only data collected by one shot of a mass spectrometer is used to obtain the analysis results of multiple protein modifications in the sample to be tested. As for peptide fragment enrichment, DDA spectrogram library construction and the like of different modified proteins, the prior art can be referred to, and is not particularly limited herein.
In one implementation manner of the present application, the method for analyzing multiple protein modifications of a biological sample further includes obtaining DDA spectra corresponding to all the peptide fragments of the protein modification type to be detected according to the enriched peptide fragments.
It should be noted that, in principle, if the DDA spectrogram library of the biological sample is obtained, the DDA spectrogram library can be directly used without repeating the building of the DDA spectrogram library according to the enriched peptide fragments; however, for a new test sample, or for a new addition of a modified type of protein to be tested, a library of DDA profiles corresponding to the enriched polypeptides is first constructed.
In one implementation of the present application, the types of protein modifications to be detected are two or more protein modifications.
It should be noted that the method of the present application is key to realize simultaneous detection of multiple protein modification types, that is, a mass spectrometer is only used to acquire data by one shot to obtain multiple protein modification analysis results; thus, the methods of the present application are particularly useful for the detection of two or more protein modifications, such as the common simultaneous detection of both phosphorylated and glycosylated protein modifications; or, detecting the modification of three proteins, namely phosphorylation protein modification, glycosylation protein modification and acetylation protein modification at the same time; or, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis. In principle, the number of protein modifications that can be simultaneously detected by the methods of the present application is not limited.
In one implementation manner of the present application, the library establishment mass spectrum acquisition parameters of the DDA spectrum library include that a Tims ion mobility mode is adopted, the scanning range is 100-1700m/z, and the ion mobility range is 0.60-1.60V.S/cm2。
In one implementation manner of the application, the library establishing mass spectrum acquisition parameters of the DDA spectrum library further comprise 1600V of ion transmission tube voltage, 3.0L/min of drying air flow rate, 180 ℃ of drying temperature, 10 parent ions selected and 10ev of fragmentation energy.
In one implementation of the present application, the DDA spectrogram library is obtained by analyzing and identifying acquired DDA mass spectrometry data with protein identification software.
Preferably, the protein identification software is Maxquant, Proteome discover or Mascot.
In one implementation of the present application, the parameters for DIA acquisition include the use of Tims ion mobility DIA-PASEP mode, the scan range 302-1077m/z, and the 1/k0 settings of 0.602-1.336V.S/cm2。
In one implementation of the present application, the parameters for DIA acquisition further include ion transport tube voltage 1600V, drying airflow rate 3.0L/min, drying temperature 180 ℃, fragmentation energy 10ev, window width 25m/z, window number of segments 4, number of windows per segment 8, PASEF cycle time 100 ms.
In an implementation manner of the present application, analyzing the acquired DIA information based on the DDA library to obtain an analysis result of protein modification of the sample to be detected specifically includes using DIA protein component analysis software, and performing DIA data analysis by using the DDA library corresponding to the peptide segment of all protein modification types to be detected as a database to obtain an analysis result of protein modification of the sample to be detected.
Preferably, the DIA proteomic analysis software is Skyline, Spectronaut, or DiaNN.
In an implementation manner of the present application, analyzing the acquired DDA information based on the DDA spectrogram library to obtain an analysis result of protein modification of the sample to be detected specifically includes analyzing the acquired DDA information by using the DDA spectrogram libraries corresponding to all peptide segments of the protein modification type to be detected by using a comparison analysis method to obtain an analysis result of protein modification of the sample to be detected.
Preferably, the alignment analysis method is a Match between runs analysis method.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
according to the method for analyzing the modification of multiple proteins in the biological sample, the enriched peptide segments of the protein modification types to be detected are mixed, and the DDA spectrogram library of each peptide segment is combined, so that the analysis results of the modification of the multiple proteins in the sample to be detected can be analyzed and obtained only by acquiring data of the mixed peptide segments once; the detection period of the modification of various proteins is shortened, the machine time of a mass spectrometer is saved, and the detection cost of the modification of various proteins is reduced.
Drawings
FIG. 1 is a prior art method of analyzing multiple protein modifications in a biological sample;
FIG. 2 is a method of analyzing multiple protein modifications in a biological sample according to an embodiment of the present application;
FIG. 3 is a method of analyzing multiple protein modifications in a biological sample modified in the examples of the present application;
FIG. 4 is another method of analyzing multiple protein modifications in a biological sample modified in the examples of the present application;
FIG. 5 is a graph showing the results of polyacrylamide gel electrophoresis of the proteins extracted in the examples of the present application.
Detailed Description
The present application will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other devices, materials, methods, etc. in various instances. In some instances, certain operations related to the present application have not been shown or described in detail in this specification in order to avoid obscuring the core of the present application from excessive description, and a detailed description of such related operations is not necessary for those skilled in the art, and the related operations will be fully understood from the description in the specification and the general knowledge of the art.
In the prior art, if a plurality of protein modifications need to be detected, as shown in fig. 1, a sample 11 is usually subjected to protein extraction 12; then carrying out protein enzymolysis 13 on the extracted protein to obtain an enzymolysis peptide segment; respectively carrying out corresponding peptide enrichment on peptide fragments subjected to protein enzymolysis digestion according to the protein modification types to be detected; for example, for detecting the modification of phosphorylated protein, phosphorylation enrichment 141 is performed to obtain phosphorylated peptide segment 142; respectively carrying out phosphorylation DDA spectrogram library establishment 1431 and phosphorylation peptide DIA acquisition 1432 according to the enriched phosphorylation peptide fragments; finally, DIA data 144 is analyzed based on DDA spectrogram library to obtain phosphorylated protein modification detection results; for another example, when detecting the modification of glycosylated protein, glycosylation enrichment 151 is performed to obtain a glycosylated peptide segment 152; respectively carrying out glycosylation DDA spectrogram library establishment 1531 and glycosylation peptide section DIA acquisition 1532 according to the enriched glycosylation peptide sections; finally, the DIA data 154 was analyzed based on DDA profile library to obtain glycosylated protein modification detection results. Thus, it can be seen that the prior art assays for multiple protein modifications require one DIA collection for each protein modification, which greatly increases assay cost and time.
The application creatively provides that the enriched peptide segments are mixed, and data acquisition is directly carried out on the mixed peptide segments by one needle, so that various protein modification analysis results can be obtained. As shown in fig. 2, similarly, a sample 21 is subjected to protein extraction 22; then carrying out protein enzymolysis on the extracted protein 23 to obtain an enzymolysis peptide segment; respectively carrying out corresponding peptide enrichment on peptide fragments subjected to protein enzymolysis digestion according to the protein modification types to be detected; for example, for detecting the modification of phosphorylated protein, phosphorylation enrichment 241 is performed to obtain phosphorylated peptide segment 242; performing glycosylation enrichment 251 to obtain a glycosylated peptide segment 252 when detecting the modification of the glycosylated protein; these steps are the same as the prior art; in contrast, according to the method of the present application, a phosphorylation DDA spectrogram library is established 243 according to the enriched phosphorylated peptide fragments, and a glycosylation DDA spectrogram library is established 253 according to the enriched glycosylation peptide fragments; then, all the peptide fragments are mixed to obtain a mixed peptide fragment 26, and the mixed peptide fragment 26 is subjected to one-time peptide fragment sample DIA collection 27; finally, the results of the detection of multiple protein modifications are obtained simultaneously based on the DIA information analysis 28 of the various repertoires, as shown in fig. 2, i.e., the results of the detection of both phosphorylated and glycosylated protein modifications are obtained simultaneously.
According to the method, when more than two modified protein groups are made simultaneously, after a spectrum library is established for each protein modification, when a sample DIA is operated, each sample can obtain more than two protein modification results only by acquiring DIA mode data once; in the traditional method, one modified protein is required to be collected once, two modified proteins are required to be collected for 2 times, and the number of the collected times is the same as that of the modified types of the detected proteins. Therefore, the more samples and the more protein modification types are analyzed at one time, the more cycle and machine time cost are saved. As shown in fig. 3, detection of acetylated protein modification is further added on the basis of the scheme shown in fig. 2, wherein protein extraction 32 is performed on a sample 31, and then protein enzymolysis 33 is performed on the extracted protein to obtain an enzymolysis peptide fragment, which are the same as the technical scheme shown in fig. 2; only increases the enrichment of the corresponding acetylated peptide fragment in the aspect of enriching the peptide fragment, and specifically, performs phosphorylation enrichment 341 to obtain a phosphorylated peptide fragment 342 aiming at the modification detection of phosphorylated protein; detecting the modification of the glycosylated protein, and performing glycosylation enrichment 351 to obtain a glycosylated peptide segment 352; detecting the modification of acetylated protein, and performing acetylation enrichment 361 to obtain an acetylated peptide fragment 362; establishing a phosphorylation DDA spectrogram library according to the enriched phosphorylation peptide fragments 343, establishing a glycosylation DDA spectrogram library according to the enriched glycosylation peptide fragments 353, and establishing an acetylation DDA spectrogram library according to the enriched acetylation peptide fragments 363; then, all the peptide fragments are mixed to obtain a mixed peptide fragment 37, and the mixed peptide fragment 37 is subjected to one-time peptide fragment sample DIA collection 38; finally, analysis 39 of the DIA information based on various repertoires, as shown in fig. 3, simultaneously obtains the results of detection of phosphorylated, glycosylated and acetylated protein modifications.
In the method of the present application, the mixed peptide fragments are collected using DIA collection mode, and in the subsequent analysis, a variety of DDA spectra libraries are used to assist in the analysis of sample DIA data. In an improved scheme, DDA acquisition can also be carried out on the mixed peptide fragment, namely, a mixed peptide fragment sample is acquired by using a DDA mode, the acquired DDA data is analyzed together with a plurality of DDA spectrogram libraries, a comparison analysis method, such as an analysis method of Match between runs, is adopted, and the DDA data of a single sample is analyzed by using the spectrogram library data, so that simultaneous identification and quantification of two or more protein modifications of a sample to be detected are realized. As shown in fig. 4, taking a case of collecting phosphorylation and glycosylation at one needle, protein extraction 42 is performed on a sample 41 in the same manner; then carrying out protein enzymolysis 43 on the extracted protein to obtain an enzymolysis peptide segment; aiming at the modification detection of the phosphorylated protein, phosphorylation enrichment 441 is carried out to obtain a phosphorylated peptide segment 442; detecting the modification of the glycosylated protein, and then performing glycosylation enrichment 451 to obtain a glycosylated peptide segment 452; carrying out phosphorylation DDA spectrogram library building 443 according to the enriched phosphorylated peptide fragments, and carrying out glycosylation DDA spectrogram library building 453 according to the enriched glycosylated peptide fragments; then, all peptide fragments are mixed to obtain a mixed peptide fragment 46, and the mixed peptide fragment 46 is subjected to one-time peptide fragment sample DDA acquisition 47, namely DDA mode acquisition; and finally, analyzing the phosphorylation spectrogram database data, the glycosylation spectrogram database data and the DDA data of the sample together, analyzing the DDA data of a single sample by adopting the DDA information analysis 48 based on the Match between run, and analyzing the DDA data of the single sample by utilizing the spectrogram database data to realize the simultaneous identification and quantification of the phosphorylation proteome and the glycosylation proteome, as shown in figure 4.
Examples
1. Protein extraction and quality control
(1) Protein extraction
6 tissue samples are taken and named A, B, C, D, E, F respectively, 50mg of each sample is weighed and transferred into a 2mL EP tube, and 600 μ L of protein lysate (7M urea, 2M thiourea, 0.2% SDS, 20mM Tris), 6 μ L of protease inhibitor Cocktail (ROCHE, 20Tablets) and 6 μ L of phosphatase inhibitor (ROCHE, 10Tablets) are added; grinding the sample into a homogenate state by using an automatic grinder, centrifuging at 25000g and 4 ℃ for 20 minutes, and taking 500 mu L of supernatant, wherein the supernatant is a protein solution; dithiothreitol (DTT) was added to the protein solution to a final concentration of 10mM, and the mixture was reacted in a water bath at 37 ℃ for 30 minutes, and then Iodoacetamide (IAM) was added to the resultant mixture to a final concentration of 55mM and reacted at room temperature for 45 minutes.
(2) Protein quantification
The protein solution is quantified by using a Bradford method, which is characterized in that when a Bradford staining solution (Coomassie brilliant blue G-250) is combined with protein under an acidic condition, the maximum absorbance wavelength is transferred from 465nm to 595nm, and the protein content and the absorbance at 595nm are in a positive line correlation relationship in a certain range; therefore, a standard curve can be made according to the light absorption value of the standard substance and the protein concentration of the standard substance, the protein concentration to be detected is quantified, and the light absorption value of the 6 samples is measured at the wavelength of 595 nm.
Sampling BSA with the concentration of 0.2 mu g/mu L according to the table 1, measuring the absorbance value of a sample with the volume of the BSA increased in gradient, and fitting the absorbance value and the sample concentration to obtain a standard curve of the absorbance value and the protein concentration:
Y＝2.001X+0.0165
wherein the correlation coefficient is R2＝0.9902，R2Is greater than0.99, which shows that the standard curve of the example meets the requirements.
TABLE 1 Standard Curve sample preparation Table
| |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Double distilled water/. |
2 | 4 | 6 | 8 | 10 | 12 | 14 | 16 | 18 | 20 |
| BSA/μL | 18 | 16 | 14 | 12 | 10 | 8 | 6 | 4 | 2 | 0 |
| Working solution/. mu.L | 180 | 180 | 180 | 180 | 180 | 180 | 180 | 180 | 180 | 180 |
The concentrations of the protein solutions of the obtained sample A, B, C, D, E, F were calculated to be 10.25. mu.g/. mu.L, 12.58. mu.g/. mu.L, 9.89. mu.g/. mu.L, 10.79. mu.g/. mu.L, 13.01. mu.g/. mu.L, and 12.11. mu.g/. mu.L, respectively.
(3) Polyacrylamide gel electrophoresis
Weighing 10 mu g of protein in each sample, adding a loading buffer with the volume of 1/3 protein solution, uniformly mixing, heating at 95 ℃ for 5min, centrifuging at 5000g for 1min, taking the supernatant, dropping the supernatant into a dropping hole of 12% SDS polyacrylamide gel, performing 80V constant voltage electrophoresis for 30min, and performing 120V constant voltage electrophoresis for 120 min; after electrophoresis, the gel was placed in a rapid staining and stripping apparatus for 10min, and the gel image was taken out for scanning.
The result of polyacrylamide gel electrophoresis is shown in fig. 5, in which KDa is the protein molecular weight, M is the quality control of 9 bands, lane 1 is sample a, lane 2 is sample B, lane 3 is sample C, lane 4 is sample D, lane 5 is sample E, and lane 6 is sample F; the electrophoresis result chart shows that the protein bands of the six protein samples are uniformly distributed, the background is clean, and the six protein samples meet the qualified standard.
2. Enzymolysis
Carrying out enzymolysis on the extracted protein solution according to the protein: adding enzyme into trypsin (Hualishi scientific, 100 UG/bottle) ═ 40:1 (mu g: mu g), reacting in a water bath kettle at 37 ℃ overnight, desalting with Strata X column (Phenomentex, TUBES-10MG/1ML) the next day, and vacuum-drying to obtain peptide segment after enzymolysis; and (4) performing quality control on the peptide fragments subjected to enzymolysis by using a mass spectrometer.
The results show that the peak types and the intensities of the six protein enzymolysis products are qualified in quality control and can be used for subsequent treatment.
3. Enrichment of
Since the enrichment procedure for each modified protein is different, this step begins a separate technical route to enrich for different modified proteins. In this example, the test sample is subjected to phosphoprotein modification and glycosylated protein modification. Therefore, the enzymatic peptide fragments of the extracted protein solution are respectively subjected to phosphorylation peptide fragment enrichment and glycosylation peptide fragment enrichment. The details are as follows:
(1) enrichment of phosphorylated peptide fragments
In this example, phosphorylation enrichment reagents were prepared according to Table 2, and High pH RP column Elution buffer was prepared according to Table 3.
TABLE 2 phosphorylation enrichment reagent Table
| Loading buffer | Wash buffer | Elution buffer1 | Elution buffer2 | |
| dd H2O | 15％ | 19％ | 83.3％ | 51.67％ |
| 100 |
80％ | 80％ | 0 | 40 |
| TFA | ||||
| 5％ | 1％ | 0 | 0 | |
| Gln (Glutamine) | 30mg/mL | 0 | 0 | 0 |
| 25% ammonia water | 0 | 0 | 16.7％ | 8.33％ |
TABLE 3 High PH RP column Elution buffer recipe
| Fraction | ACN(％) | ACN(μL) | 0.1％Triethylamine(μL) |
| 1 | 5 | 50 | 950 |
| 2 | 7 | 70 | 930 |
| 3 | 8 | 80 | 920 |
| 4 | 9 | 90 | 910 |
| 5 | 10 | 100 | 900 |
| 6 | 11 | 110 | 890 |
| 7 | 13 | 130 | 870 |
| 8 | 15 | 150 | 850 |
| 9 | 17 | 170 | 830 |
| 10 | 20 | 200 | 800 |
| 11 | 25 | 250 | 750 |
| 12 | 50 | 500 | 500 |
Diluting 1.2mg of peptide fragment to 1.0 mu g/mu L by using Loading buffer for later use; as sample TiO2Weighing TiO 1:42Into TiO2Adding 1mL of Loading buffer, shaking at room temperature for 10min, centrifuging at 12000g for 30s, discarding the supernatant, and retaining TiO2Precipitating; to the above TiO2Adding diluted peptide segment sample, oscillating at 37 deg.C 1100rpm for 1 hr in constant temperature oscillator, and phosphorylating peptide segment and TiO2Binding, i.e. enrichment reaction; centrifuging the shaken sample at 12000g for 30s, collecting the supernatant to another centrifuge tube, and simultaneously keeping the precipitate; adding 1mL of Loading buffer into the precipitate, overturning the disc at room temperature for 5min, centrifuging at 12000g for 30s, and discarding the supernatant; adding 1mL of Wash buffer, turning the disc at room temperature for 5min, centrifuging at 12000g for 30s, discarding the supernatant, and repeating the step for 4 times; adding 600 μ L of Elution buffer 1, shaking at room temperature and 1200rpm for 20min, centrifuging at 12000g for 30s, and collecting the supernatant; adding 500 μ L of Elution buffer 2, shaking at room temperature and 1200rpm for 20min, centrifuging at 12000g for 30s, and collecting the supernatant; and (4) combining the supernatants collected twice, freezing and drying by suction to obtain the enriched phosphorylated peptide fragment.
(2) Enrichment of glycosylated peptide fragments
Dissolving the peptide fragment by 300 mu L of 60% ACN/0.1% FA solution, and sufficiently shaking for dissolving for later use; enrichment and fractionation was performed after column equilibration using HILIC columns (Merck, 5 μm, 150 x 4.6mm) with buffer A of 80% ACN/0.1% FA and buffer B of 0.1% FA. Collecting one tube per minute after fraction is received at 54min from 30min, collecting 24 tubes and marking; put into a freeze-pump for pumping, and then a total of 50. mu.L of 50mM HH was applied to each adjacent 4 tubes4HCO3Re-dissolving the mixture into one tube successively, and finally dividing the tube into 6 components; adding 2.5 μ L PNGase F (Biolabs, P0710S) into each component for desugarization, shaking, mixing, centrifuging, and water-bathing at 37 deg.C overnight; and (4) putting the mixture into a freezing and drying machine for drying the mixture in the next day to obtain the enriched glycosylated peptide section.
4. Separation of phosphorylated peptide fragment components
Mixing the enriched phosphorylated peptide fragments of each sample, and then re-dissolving the mixture by 300 mu L of 0.1% TFA; using a High PH RP column (Thermo Scientific Pierce)TM) And (3) component separation:
(1) activation and equilibration of High PH RP column:
A. taking out the column, centrifuging for 2min at 5000g to remove the remaining liquid in the column;
B. adding 300 μ L ACN 5000g into the column, centrifuging for 2min, and washing for 2 times;
C. add 300. mu.L of 0.1% TFA 5000g to the column and centrifuge for 2min to wash 2 times;
(2) carrying out sample loading: adding a redissolved sample (300. mu.L) to the column, centrifuging at 3000g for 2min, and retaining FT solution (FT) in another centrifuge tube;
(3) washing: adding 300 μ L water, centrifuging at 3000g for 2min, and keeping wash solution (WT) in another centrifuge tube;
(4) and (3) elution: eluting sequentially with Elution buffers prepared in Table 3, respectively labeled as F1 and F2 … F12, adding 300 μ L of Eution buffer during Elution, centrifuging at 3000g for 2min, and collecting each eluate;
(5) eluent is pressed
F1+ F7 "," F2+ F8 "," F3+ F9 "," F4+ F10 "," F5+ F11 "," F6+ F12+ FT + WT "are combined into 6 components, and the components are stored at-20 ℃ for standby after being dried.
5. Mass spectrum computer
And (3) collecting 1-hour mass spectrum data of each of the samples after the components are separated by using a tandem mass spectrometer (timsTOF Pro) in a DDA mode, mixing the phosphorylation enrichment peptide section and the glycosylation enrichment peptide section corresponding to each sample, and collecting 1-hour mass spectrum data in a DIA mode.
(1) Conditions of liquid phase separation
A self-contained C18 column (75 μm internal diameter, 1.8 μm column size,
about 25cm column length), Buffer a: h2O+0.1％FA，Buffer B：ACN+0.1％FA
The liquid phase gradient is shown in table 4.
TABLE 4 liquid phase gradient
| Time (min) | Flow rate (μ L/min) | B％ |
| 0 | 0.3 | 2 |
| 45 | 0.3 | 22 |
| 50 | 0.3 | 35 |
| 55 | 0.3 | 80 |
| 60 | 0.3 | 80 |
(2) DDA library construction mass spectrum acquisition parameter
Adopting Tims ion mobility mode, the scanning range is 100-1700m/z, and the ion mobility range is 0.60-1.60V.S/cm2。
The voltage of an ion transmission tube is 1600V, the flow rate of drying air is 3.0L/min, the drying temperature is 180 ℃, 10 parent ions are selected, and the fragmentation energy is 10 ev.
(3) DIA detection of mass spectrometry acquisition parameters
The scanning range 302-1077m/z, 1/k0 is set to be 0.602-1.336V.S/cm by adopting Tims ion mobility DIA-PASEP mode2。
The voltage of an ion transmission tube is 1600V, the flow rate of drying air is 3.0L/min, the drying temperature is 180 ℃, the fragmentation energy is 10ev, the window width is 25m/z, the number of window segments is 4, the number of windows in each segment is 8, and the PASEF cycle time is 100 ms.
Creation of DDA data
And (3) respectively analyzing and identifying the DDA mass spectrum data of the phosphorylated peptide segment and the DDA mass spectrum data of the glycosylated peptide segment by using protein identification software, such as Maxquant, Proteome resolver, Mascot and the like to respectively obtain a phosphorylated Proteome spectrogram library and a glycosylated Proteome spectrogram library. In this example, Maxquant software is specifically used for analysis and identification to obtain a DDA spectrogram library. The number of spectrogram library identifications is shown in Table 5.
TABLE 5 identification number of DDA spectrogram libraries of phosphorylated proteomes and glycosylated proteomes
| Type of modification | Number of all peptide fragments | Number of modified peptide fragments | Number of modified proteins |
| Library of phosphorylated protein profiles | 47826 | 19648 | 5608 |
| Library of glycosylated protein profiles | 94912 | 11845 | 4205 |
7. Data analysis
And (3) using DIA protein component analysis software, such as Skyline, Spectronaut, DiaNN and other software, and taking the mixed modified proteome spectrogram library obtained by mixing the phosphorylated proteome spectrogram library and the glycosylated proteome spectrogram library as a database to analyze DIA data and obtain the identification and quantitative analysis results of the phosphorylated proteome and the glycosylated proteome.
This example specifically uses Spectronaut software for DIA data analysis, and the results are shown in Table 6.
TABLE 6 DIA Collection analysis of phosphorylated and glycosylated peptide fragments
| Sample(s) | Number of phosphorylated peptide fragments | Number of phosphorylated proteins | Number of glycosylated peptide segments | Number of glycosylated proteins |
| A | 15221 | 4025 | 7646 | 2747 |
| B | 14325 | 3968 | 7494 | 2676 |
| C | 13537 | 3869 | 7538 | 2682 |
| D | 15860 | 4149 | 8027 | 2866 |
| E | 15863 | 4175 | 7965 | 2835 |
| F | 16096 | 4174 | 8083 | 2856 |
In addition, the collection of the phosphorylated and glycosylated protein groups DIA data was performed according to the conventional method, and the results are shown in Table 7.
TABLE 7 results of analysis of phosphorylated proteomes and glycosylated proteomes by conventional methods
| Sample(s) | Number of phosphorylated peptide fragments | Number of phosphorylated proteins | Number of glycosylated peptide segments | Number of glycosylated proteins |
| A | 16440 | 4347 | 8366 | 2967 |
| B | 16071 | 4289 | 8089 | 2886 |
| C | 15621 | 4179 | 8141 | 2895 |
| D | 17129 | 4481 | 8663 | 3095 |
| E | 17132 | 4498 | 8602 | 3065 |
| F | 17384 | 4508 | 8729 | 3081 |
The results in tables 6 and 7 show that the number of phosphorylated peptide segments and glycosylated peptide segments detected by the method is higher, and is only slightly lower than the number of the results of the traditional method, and the method is within the error range of experimental analysis. Therefore, the method can completely meet the market and scientific research requirements; in addition, various detection periods are greatly shortened, the mass spectrometer is saved, and the detection cost is reduced.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (10)
1. A method for analyzing multiple protein modifications in a biological sample, comprising: respectively carrying out corresponding peptide enrichment on peptide fragments subjected to protein enzymolysis digestion according to protein modification types to be detected; the peptide segment of the protein enzymolysis digestion is the peptide segment obtained by carrying out enzymolysis digestion on the protein extracted from a sample to be detected; mixing the enriched peptide fragments to obtain a protein modification type mixed peptide fragment to be detected; performing one-time DIA collection and/or DDA collection on the mixed peptide fragments; and analyzing the acquired DIA information and/or DDA information based on a DDA spectrogram library corresponding to the peptide fragment of the protein modification type to be detected to obtain an analysis result of the protein modification of the sample to be detected.
2. The method of claim 1, wherein: and further comprising respectively obtaining DDA spectrogram libraries corresponding to the peptide fragments of all the protein modification types to be detected according to the enriched peptide fragments.
3. The method of claim 1, wherein: the protein modification types to be detected are two or more protein modifications.
4. A method according to any one of claims 1-3, characterized in that: the library establishing mass spectrum acquisition parameters of the DDA spectrum library comprise that a Tims ion mobility mode is adopted, the scanning range is 100-1700m/z, and the ion mobility range is 0.60-1.60V.S/cm2。
5. The method of claim 4, wherein: the acquisition parameters of the library-building mass spectrum of the DDA spectrogram library further comprise 1600V of ion transmission tube voltage, 3.0L/min of drying air flow rate, 180 ℃ of drying temperature, 10 parent ions selected and 10ev of fragmentation energy.
6. The method of claim 5, wherein: the DDA spectrogram library is obtained by analyzing and identifying acquired DDA mass spectrum data by protein identification software;
preferably, the protein identification software is Maxquant, protome resolver or Mascot.
7. A method according to any one of claims 1-3, characterized in that: the parameters of DIA acquisition comprise that Tims ion mobility DIA-PASEP mode is adopted, the scanning range is 302-1077m/z, and 1/k0 is set to be 0.602-1.336V.S/cm2。
8. The method of claim 7, wherein: the DIA parameters also included ion transport tube voltage 1600V, drying gas flow rate 3.0L/min, drying temperature 180 deg.C, fragmentation energy 10ev, window width 25m/z, window number 4, window number per segment 8, and PASEF cycle time 100 ms.
9. A method according to any one of claims 1-3, characterized in that: analyzing the acquired DIA information based on the DDA spectrogram library to obtain an analysis result of the protein modification of the sample to be detected, wherein DIA data analysis is performed by using DIA protein component analysis software and taking the DDA spectrogram libraries corresponding to all the peptide fragments of the protein modification type to be detected as a database to obtain an analysis result of the protein modification of the sample to be detected;
preferably, the DIA proteomics analysis software is Skyline, Spectronaut, or DiaNN.
10. A method according to any one of claims 1-3, characterized in that: analyzing the acquired DDA information based on the DDA spectrogram library to obtain an analysis result of protein modification of the sample to be detected, and specifically, analyzing the acquired DDA information by using the DDA spectrogram libraries corresponding to all the peptide segments of the protein modification type to be detected by adopting a comparison analysis method to obtain an analysis result of protein modification of the sample to be detected;
preferably, the alignment analysis method is a Match between runs analysis method.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104792894A (en) * | 2015-04-21 | 2015-07-22 | 首都医科大学附属北京儿童医院 | Protein characteristic spectrum of active tuberculosis in children and method for creating protein characteristic spectrum |
| CN111024873A (en) * | 2019-12-26 | 2020-04-17 | 上海鹿明生物科技有限公司 | Method for establishing and quantifying phosphorylated proteome database in whole growth period of rice |
| CN111435138A (en) * | 2019-01-15 | 2020-07-21 | 中国科学院分子细胞科学卓越创新中心 | Pancreatic cancer protein biomarker and application thereof |
| CN111537659A (en) * | 2020-06-03 | 2020-08-14 | 北京航空航天大学 | Method for screening biomarkers |
| CN113484449A (en) * | 2021-07-14 | 2021-10-08 | 深圳华大基因股份有限公司 | Method for high-throughput quantitative and qualitative analysis of protein |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104792894A (en) * | 2015-04-21 | 2015-07-22 | 首都医科大学附属北京儿童医院 | Protein characteristic spectrum of active tuberculosis in children and method for creating protein characteristic spectrum |
| CN111435138A (en) * | 2019-01-15 | 2020-07-21 | 中国科学院分子细胞科学卓越创新中心 | Pancreatic cancer protein biomarker and application thereof |
| CN111024873A (en) * | 2019-12-26 | 2020-04-17 | 上海鹿明生物科技有限公司 | Method for establishing and quantifying phosphorylated proteome database in whole growth period of rice |
| CN111537659A (en) * | 2020-06-03 | 2020-08-14 | 北京航空航天大学 | Method for screening biomarkers |
| CN113484449A (en) * | 2021-07-14 | 2021-10-08 | 深圳华大基因股份有限公司 | Method for high-throughput quantitative and qualitative analysis of protein |
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