CN110133170B - Protein detection method for complex sample - Google Patents
Protein detection method for complex sample Download PDFInfo
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- CN110133170B CN110133170B CN201910351754.4A CN201910351754A CN110133170B CN 110133170 B CN110133170 B CN 110133170B CN 201910351754 A CN201910351754 A CN 201910351754A CN 110133170 B CN110133170 B CN 110133170B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—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
- G01N2030/8813—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
- 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
The invention provides a protein detection method for a complex sample, which comprises the following steps: carrying out chromatographic separation and mass spectrum scanning on the complex sample to acquire mass spectrum data, and during the mass spectrum scanning and acquiring the mass spectrum data, adopting a DDA mode to scan and acquire and insert PRM mode scanning and acquisition; and carrying out qualitative and quantitative analysis on the protein in the mass spectrum data to obtain the expression information of the full-spectrum protein of the complex sample. By adjusting the acquisition modes of the two existing mass spectrum data, the PRM acquisition mode is inserted in the DDA acquisition process to more accurately quantify the target protein, and the full-spectrum protein expression condition of the sample and more accurate quantification of the target protein are obtained within a shorter time range. The method has the advantages of high throughput of full-spectrum coverage of protein in a DDA acquisition mode and high sensitivity and accuracy of targeted quantification in a PRM acquisition mode, and is simple to operate, less in time consumption and short in experiment period.
Description
Technical Field
The invention relates to the field of protein expression quantity detection, in particular to a protein detection method for a complex sample.
Background
Data Dependent Acquisition (DDA) is the most common mass spectrometry Data acquisition mode at present, and does not need to preset ion information or establish a spectrogram Data set, so that the application process is convenient and rapid. Different ions are subjected to fragmentation scanning according to the real-time signal intensity of the detection sample, and the completeness of ion species in the detection sample can be ensured to a certain extent. But ion collection is carried out according to the real-time signal intensity, so that some ions with lower abundance have the phenomenon of lower credibility or loss in the quantitative process.
Parallel Reaction Monitoring (PRM) belongs to mass-spectrometric targeted analysis, and the entire fragment ion spectrum of each target parent ion is continuously recorded during the whole liquid phase separation process. In contrast to the mode in which a conventional SRM detects only target ion pairs, the PRM detects all fragment information within a selected parent ion window. The main advantages of PRM are the use of an ultra-high resolution Orbitrap mass analyser, which can distinguish interference information from the real signal and better guarantee the selectivity of the analysis compared to the conventional SRM method. However, the target detection of thousands of proteins cannot be completed in a short time.
At present, under the condition that the complexity of proteins in complex samples such as tissues, cells and body fluids is high and the abundance of target peptide fragments in the samples is low, no effective solution is available for rapidly and efficiently obtaining the expression quantity data of full-spectrum proteins of the samples.
Disclosure of Invention
The invention mainly aims to provide a protein detection method for a sample containing complex protein, so as to solve the problem that the full-spectrum protein expression detection in the complex sample cannot be completed in a short time in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for detecting a protein in a complex sample, the method comprising: performing chromatographic separation and mass spectrum scanning on the complex sample to acquire mass spectrum data, wherein in the process of acquiring the mass spectrum data by mass spectrum scanning, PRM mode scanning acquisition is inserted in the process of DDA mode scanning acquisition; and carrying out qualitative and quantitative analysis on the protein in the mass spectrum data to obtain the expression information of the full-spectrum protein of the complex sample.
Further, in a complex sample, scanning and collecting a PRM mode in a time window corresponding to a target peptide segment of the target protein; preferably, the DDA mode scan and the scan acquisition of the PRM mode are performed using a quadrupole mass spectrometer or a triple spectrometer.
Further, the step of analyzing the protein quantification in the mass spectrometric data comprises: carrying out quantitative analysis on mass spectrum data acquired by a DDA mode according to the peak area of the primary chromatogram; mass spectral data acquired using PRM mode was quantitatively analyzed based on the sum of secondary fragment ions targeting the top three high of the peptide fragment.
Further, the complex sample is chromatographed using a chromatography column, wherein the chromatography column comprises: the outer diameter of the chromatographic column tube is 355-365 mu m, and the inner diameter of the chromatographic column tube is 100-200 mu m; the filler is C18 filler and is filled in the chromatographic column tube; and a chromatographic column tip integrally designed with the chromatographic column tube.
Further, the length of the chromatographic column tube is 150-300 mm.
Further, the inner diameter of the chromatographic column tip (the inner diameter of the most pointed end position of the chromatographic column tip) is 2-8 μm, and preferably 3-6 μm; preferably, the particle size of the filler is 1.5 to 3 μm, preferably 1.8 to 2.5 μm.
Further, in the step of chromatographic separation, a chromatographic separation gradient of 75-150 min is adopted for separation.
Further, the complex sample is a tissue sample, a cell sample, or a body fluid sample.
Further, the complex sample is a plant sample, an animal sample or a microorganism sample.
Further, the target protein is one or more proteins in the mass spectrum historical data acquired based on the DDA mode.
By applying the technical scheme of the invention, the existing two mass spectrum data acquisition modes are adjusted, the protein with relatively high expression abundance in a complex sample can be quantified only through the DDA acquisition mode, and the protein with low expression abundance or the interested target protein is quantified through the PRM acquisition mode, so that the full-spectrum protein expression condition of the sample can be obtained through the DDA mode, the PRM acquisition mode can be alternated to more accurately quantify the target protein in the DDA acquisition process, and the full-spectrum protein expression condition of the sample and more accurate quantification condition of the target protein can be obtained basically in a shorter time range spent in the DDA acquisition mode. Namely, the method has the advantages of high throughput of full-spectrum coverage of the protein in the DDA acquisition mode and high sensitivity and accuracy of targeted quantification in the PRM acquisition mode, and is simple to operate, less in time consumption and short in experimental period.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
Interpretation of terms:
complex samples: samples of tissues, cells or body fluids including animals, plants, microorganisms and the like, and samples capable of reaching a protein detection depth covering thousands of times are called complex samples. The existing method can detect thousands of proteins even by using more mass spectrometry machines for the samples, but the proteins with lower expression abundance can still not be detected.
History data: refers to mass spectrum data of the same complex sample acquired based on previous DDA mode scanning before the operation of 'scanning acquisition of alternating PRM mode in the process of scanning acquisition by DDA mode' of the application is carried out. Based on the previously employed mass spectral data, some of the target proteins of interest or interest in the complex sample may be known. Furthermore, when the operation of "scan acquisition in which the PRM mode is interspersed in the process of scan acquisition in the DDA mode" in the present application is performed, the relevant information (for example, a time window) of the target protein acquired by the PRM can be accurately set.
As mentioned in the background art, there are many cases in the prior art that require the detection of the entire protein expression pattern and/or expression level of a sample, but in the existing protein mass spectrometry detection method, either the protein mass spectrometry data acquisition is performed based on the DDA scan pattern, or the target protein of interest is quantified in the case of known sample expression profiles, so that the mass spectrometry data acquisition is performed only in the PRM data acquisition mode. However, when the complexity of the protein in the sample to be detected is relatively high, on one hand, the DDA data collection mode takes a long time to separate the complex proteins as much as possible, but some low-abundance protein peptides may not be detected, and thus quantification is not performed. The PRM mode can be used for more accurate quantification, but the time is longer, and the expression of all proteins in a sample containing complex proteins cannot be realized in a relatively short time.
In order to improve the above situation in the prior art, the inventor adjusts the two acquisition modes of mass spectrum data, and can quantify a protein with relatively high expression abundance in a complex sample only through the DDA acquisition mode, and quantify a protein with low expression abundance or a target protein of interest through the PRM acquisition mode, so that the full-spectrum protein expression condition of the sample can be obtained through the DDA mode, and the target protein can be quantified more accurately through the PRM acquisition mode in the DDA acquisition process, and the full-spectrum protein expression condition of the sample and the more accurate quantification condition of the target protein can be obtained basically in a shorter time range spent in the DDA acquisition mode. Namely, the method has the advantages of high throughput of full-spectrum coverage of the protein in the DDA acquisition mode and high sensitivity and accuracy of targeted quantification in the PRM acquisition mode, and is simple to operate, less in time consumption and short in experimental period.
Therefore, on the basis of the above research results, in an exemplary embodiment of the present application, a method for detecting a protein in a complex sample is provided, the method comprising: performing chromatographic separation and mass spectrum scanning on the complex sample to acquire mass spectrum data, wherein in the process of acquiring the mass spectrum data by mass spectrum scanning, PRM mode scanning acquisition is inserted in the process of DDA mode scanning acquisition; and carrying out qualitative and quantitative analysis on the protein in the mass spectrum data to obtain the expression information of the full-spectrum protein of the complex sample.
As described above, the protein detection method for such complex protein-containing samples of the present application has the advantages of high throughput of full-spectrum coverage of protein in DDA acquisition mode and high sensitivity and accuracy of targeted quantification in PRM acquisition mode by adjusting the acquisition mode of mass spectrum data, i.e., performing PRM acquisition mode in a specific time window during DDA acquisition mode, based on the existing mass spectrum protein detection method.
The above PRM acquisition mode may be set in different time windows during the DDA mode according to different research purposes. In a preferred embodiment, the time window corresponding to the target peptide fragment of the target protein in the complex sample is scanned and acquired in PRM mode. For example, protein extraction is performed on seeds of different development stages of rice to obtain the expression condition of proteins affecting the nutritional quality or taste of rice, and then PRM acquisition can be set according to the ion information (including retention time, m/z ratio and the like) of the targeted peptide fragment of the gluten in the DDA acquisition process. That is, in the DDA collection process, with the adoption of different protein peptide information, when the peptide information of gluten appears, the acquisition is performed in the PRM mode, and after the peptide information of gluten is collected, the DDA mode is collected. It should be noted here that, the acquisition in the PRM mode instead does not mean that the DDA acquisition mode is stopped, but the DDA acquisition mode is squeezed by the PRM acquisition mode in the same time window, and the time of the data acquired by the PRM mode occupies the time of the data acquired by the DDA mode, so that other proteins that should be acquired by the DDA mode in the time window may be missed.
In order to make the detection result relatively more accurate, in the preferred embodiment of the present application, a quadrupole mass spectrometer or a triple-junction mass spectrometer is used for performing DDA mode scanning and PRM mode scanning acquisition. The four-rod mass spectrometer or the three-in-one mass spectrometer is a high-resolution mass spectrometer, the collected mass spectrum data is more accurate, and particularly, the low-abundance protein is improved, so that the subsequent analysis result is more accurate, and the detection result reliability is higher. Specifically, a nanoliter chromatograph Easy-nLC, a high-resolution mass spectrometer Q-active series, and a seemer fly three-in-one high-resolution mass spectrometer Orbitrap Fusion, Lomus and the like can be adopted.
In the above methods provided by the present application, the method for quantifying protein from data collected in two collection modes may be performed according to the existing method. In a preferred embodiment of the present application, the step of quantitatively analyzing the protein in the mass spectrometric data comprises: carrying out quantitative analysis on mass spectrum data acquired by a DDA mode according to the peak area of the primary chromatogram; mass spectral data acquired using PRM mode was quantitatively analyzed based on the sum of secondary fragment ions targeting the top three high of the peptide fragment.
In order to further improve the protein separation degree of a sample containing complex proteins and further obtain better mass spectrum acquisition data, the applicant also improves the conventional chromatographic column for protein detection, and the improved chromatographic column has better protein separation degree. In a preferred embodiment of the present application, a chromatography column is used for performing chromatographic separation of a complex sample, wherein the chromatography column comprises: the outer diameter of the chromatographic column tube is 355-365 mu m, and the inner diameter of the chromatographic column tube is 100-200 mu m; the filler is C18 filler and is filled in the chromatographic column tube; and a chromatographic column tip integrally designed with the chromatographic column tube.
According to the chromatographic column in the preferred embodiment, the chromatographic column tip integrally designed with the chromatographic column tube is adopted, and the inner diameter of the chromatographic column is expanded to 100-200 μm from 75 μm commonly used for protein separation, so that the inner diameter of the chromatographic column is relatively wide, and the wide inner diameter can ensure that the column pressure is maintained within the normal pressure range (within 280 bar) of a non-high pressure resistant conventional liquid phase system when the chromatographic elution flow rate is increased, thereby improving the chromatographic stability; meanwhile, the method is beneficial to increasing the sample loading amount of the sample and improving the sensitivity of mass spectrum detection. In addition, most of the existing chromatographic columns are flat-head chromatographic columns matched with a chromatographic spray needle, namely, the chromatographic columns do not have column tips, and the elution peaks of the chromatographic columns have trailing phenomena in the elution process, so that the column efficiency is relatively poor. The improved integrally formed chromatographic analysis column has no tailing phenomenon during elution and has better protein separation degree. Therefore, the chromatographic analysis column improved by the method has better peptide fragment separation degree and higher stability, and is further more favorable for carrying out quantitative analysis on the target protein, so that the quantitative result is more accurate.
The improved chromatographic column enlarges the inner diameter of a chromatographic column tube on one hand, and improves the structure of a chromatographic column tip on the other hand, so that a peptide segment is separated, and the separation effect is better. In addition, in order to further improve the separation effect of the protein, the length of the chromatographic column tube is further increased on the basis of expanding the inner diameter width, and in a preferred embodiment of the present application, the length of the chromatographic column tube is 150-300 mm.
The length of the chromatographic column can be selected within the above range according to the abundance of proteins in the sample to be separated and the purpose of the study. For example, the length of a chromatographic column tube with the length of 150mm or 300mm can be selected, and the separation effect of the peptide fragment can be better by matching with the gradient separation duration of 75-150 min.
In the improved chromatographic analysis column, the chromatographic column tip integrally formed with the chromatographic column tube is obtained by stretching one end of the chromatographic column tube, along with the stretching, the inner diameter of the column tube is gradually reduced, the thickness of the inner diameter and the outer diameter is gradually reduced, the inner diameter and the outer diameter of the formed chromatographic column tip gradually tend to approach, at the moment, the inner diameter of the chromatographic column tip (namely the inner diameter of the opening at the most pointed end of the chromatographic column tip) is 2-8 μm, the optimal inner diameter is 3-6 μm according to different actual samples, the inner diameter is too large, the inner diameter is not beneficial to spraying, the inner diameter is too small, the pressure of the chromatographic column tube is easily too large, and the operation stability of the chromatographic column tube is affected.
The chromatographic column mentioned in the present application can adopt similar parameters to those of the existing chromatographic column except for the inner diameter and the integrally formed chromatographic column tip, and other parameters such as the packing or the outer diameter. In order to more effectively and efficiently separate the protein in the synovial fluid, in a preferred embodiment of the present invention, the filler in the chromatographic column is a C18 filler, and the particle size of the filler is preferably 1.5 to 3 μm, and more preferably 1.8 to 2.5 μm. Selecting a C18 packing with a particle size within this range has the advantage of high chromatographic resolution while the pressure in the chromatographic system is low.
The method for detecting the protein expression provided by the application is different from samples containing complex proteins according to different actual research projects. In the present application, it may be a plant sample, an animal sample or a microorganism sample.
The target protein in the above method may also be different according to the actual application field or scene. In a preferred embodiment of the present application, the protein of interest is one or more proteins in the mass spectral historical data acquired based on the DDA mode.
The advantageous effects of the present application will be further described with reference to specific examples.
Example 1: detection of protein markers in liver tissue samples
1. Taking 9 liver tissue samples, taking 10 mu g of peptide fragments of the liver tissue after proteolysis of each sample, adding 25 mu L of loading buffer solution, shaking and uniformly mixing to prepare a tumor sample peptide fragment solution for later use;
2. putting 5 mu L of the liver sample peptide fragment solution prepared in the step 1 on a 96-hole sample loading plate for chromatographic sample injection;
3. the chromatographic separation gradient settings are as follows in table 1:
mobile phase A: 100% chromatographic grade water +0.2 v% formic acid;
mobile phase B: 80% chromatographic grade acetonitrile +0.2 v% formic acid;
chromatographic gradient: gradient for 150 min.
Table 1:
| Time | duration of time | Flow rate (nanoliter/minute) | B％ |
| 0 | 0 | 600 | 5 |
| 140 | 140 | 600 | 45 |
| 141 | 1 | 600 | 95 |
| 150 | 9 | 600 | 95 |
4. The mass spectrometer Q-active HF (Thermo) was set as follows:
primary mass spectrum, scanning range of 300-1400m/z, resolution of 120000(@ m/z 200), AGC target (automatic gain target) of 3e6, Maximum IT (Maximum injection time) of 80 ms;
secondary scan, Isolation window 1.6Da, resolution 15000(@ m/z 200), AGC target automatic gain target 2e4, Maximum injection time IT 20MS, Microscan 1, MS2Activation Type HCD, NCE 27; the number of secondary scans Top 20.
During the DDA scan, 10 PRM scan events are interspersed, PRM scan peptide segments include the list in Table 2, Isolation window:1.6Da, resolution: 15000(@ m/z 200), AGC target:2e4, MaximuIT: 40MS, Microscan: 1, MS2Activation Type: HCD (high energy collision mode), NCE (collision energy): 27.
Table 2:
5. and (3) performing Proteome resolver processing on the mass spectrum data, and then performing Skyline software processing on the mass spectrum data to calculate the expression amount of the protein marker in each tissue sample. The results are shown in tables 3 and 4:
table 3:
| sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | |
| DDA_PRO_ID | 4395 | 4401 | 4368 | 4289 | 4374 | 4427 | 4364 | 4386 | 4251 |
| DDA+PRM_PRO_ID | 4078 | 4071 | 4035 | 3927 | 4037 | 4101 | 4070 | 4090 | 3950 |
Table 4:
| target protein | Number of DDA _ spectrogram | Number of DDA + PRM spectrum |
| NFIA | 0 | 68 |
| SMARCA5 | 0 | 95 |
| AHCTF1 | 0 | 99 |
| CREB3L3 | 0 | 220 |
| HHEX | 2 | 21 |
| GATAD2B | 3 | 64 |
| ARRB2 | 7 | 37 |
| ZHX3 | 8 | 102 |
| DR1 | 11 | 292 |
| CADM1 | 11 | 546 |
| LAMP1 | 14 | 253 |
| DNAJC1 | 16 | 262 |
| NFIC | 17 | 226 |
| SERPINB9 | 17 | 242 |
| CLEC2D | 19 | 236 |
| PDS5B | 19 | 420 |
| TAP1 | 24 | 393 |
| CEACAM1 | 27 | 506 |
| MTA2 | 33 | 274 |
| CRK | 43 | 369 |
| HMGN5 | 50 | 1069 |
| SMARCC2 | 66 | 577 |
| MLXIPL | 74 | 369 |
| PTPN6 | 82 | 713 |
| STAT5B | 91 | 241 |
| STAT1 | 91 | 284 |
| AP1G1 | 101 | 671 |
| YBX1 | 137 | 1991 |
| LGALS9 | 144 | 706 |
| STAT3 | 154 | 573 |
| PRDX1 | 416 | 1317 |
As can be seen from tables 3 and 4, the number of full-spectrum proteins detected by the method of the present application has no significant effect compared to the conventional protein detection method (the slightly smaller specific value is because the DDA mode cannot be acquired at the same time when the mass spectrum data is acquired in the PRM mode, and thus there is a slight loss in protein detection during that time). By inserting the PRM acquisition mode, the detection method can detect proteins which cannot be detected in the DDA mode, can obviously improve the detection times of low-abundance proteins in the DDA mode, and improves the detection sensitivity and the quantitative accuracy in the detection time same as that of the conventional method.
Example 2: full-spectrum detection of clinical paraffin section sample of gastric cancer and detection of molecular typing target protein marker
1. Adding 10 mu g of peptide fragments of paraffin section sample after proteolysis into 25 mu L of sample loading buffer solution, shaking and uniformly mixing to prepare a tumor sample peptide fragment solution for later use;
2. putting 5 mu L of the peptide fragment solution of the paraffin section sample prepared in the step 1 on a 96-hole sample loading plate for chromatographic sample injection;
the chromatographic separation gradient settings are as follows in table 5:
mobile phase A: 100% chromatographic grade water + 0.2V% formic acid;
mobile phase B: 80% chromatographic grade acetonitrile + 0.2V% formic acid;
chromatographic gradient: gradient 75 min.
Table 5:
| Time | duration of time | Flow rate (nanoliter/minute) | B％ |
| 0 | 0 | 600 | 8 |
| 6 | 16 | 600 | 16 |
| 51 | 35 | 600 | 30 |
| 66 | 15 | 600 | 42 |
| 67 | 1 | 600 | 95 |
| 75 | 8 | 600 | 95 |
3. The mass spectrometer Orbitrap Fusion Lumos (Thermo) was set up as follows:
first-order mass spectrum, scanning range 300-1400m/z, resolution 120000(@ m/z 200), RF lens (%): 45, AGCtarget:5e5, Maximum IT:50 ms;
secondary scanning, Isolation window:1.6Da, MS2Activation Type: HCD, NCE: 32; a detector: ion trap, ion trap scanning mode: rapid, AGC target:5e3, Maximum IT:35ms, Microscan: 1.
During the DDA scan, 10 PRM scan events were interspersed, Isolation window:1.6Da, resolution: 15000(@ m/z 200), AGC target:1e5, Maximum IT:80MS, Microscan: 1, MS2 ActivateType: HCD, NCE: 32.
The target quantitative inclusionlist settings for the protein of interest are shown in table 6:
table 6:
4. the mass spectral data was subjected to protome scanner processing followed by Skyline software processing.
The total amount of protein detected in the mid-cancer tissue and the para-cancer tissue is shown in table 7, and the relative content values of the protein markers calculated in the cancer tissue and the para-cancer tissue in different paraffin section samples are shown in table 8.
Table 7:
| sample numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Number of oncoproteins | 2123 | 1944 | 2286 | 2278 | 2540 | 1993 | 1839 |
| Number of paratubulin | 2223 | 2267 | 2227 | 1969 | 2709 | 2688 | 2017 |
Table 8:
| cancer/Normal tissue | No.1 | No.2 | No.3 | No.4 | No.5 | No.6 | No.7 |
| RBM39 | 1.14 | 1.73 | 1.61 | 1.71 | 1.21 | 4.41 | 0.71 |
| RPL4 | 0.96 | 1.07 | 1.73 | 1.05 | 0.8 | 2.96 | 0.57 |
| SLC25A12 | 0.34 | 0.45 | 1.36 | 9.36 | 0.36 | 1.83 | 0.47 |
| HADHB | 0.32 | 0.32 | 1.43 | 0.56 | 0.33 | 1.53 | 0.44 |
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: by adjusting the acquisition modes of the two existing mass spectrum data, the protein with relatively high expression abundance in a complex sample can be quantified only through the DDA acquisition mode, and the protein with low expression abundance or the target protein of interest can be quantified through the PRM acquisition mode, so that the full-spectrum protein expression condition of the sample can be obtained through the DDA mode, and the target protein can be quantified more accurately through the PRM acquisition mode in the DDA acquisition process. Namely, the method has the advantages of high throughput of full-spectrum coverage of the protein in the DDA acquisition mode and high sensitivity and accuracy of targeted quantification in the PRM acquisition mode, and is simple to operate, less in time consumption and short in experimental period.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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1 5 10
<210> 16
<211> 30
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 16
Arg Pro Gln Tyr Ser Asn Pro Pro Val Gln Gly Glu Val Met Glu Gly
1 5 10 15
Ala Asp Asn Gln Gly Ala Gly Glu Gln Gly Arg Pro Val Arg
20 25 30
<210> 17
<211> 19
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 17
Gly Ala Glu Ala Ala Asn Val Thr Gly Pro Gly Gly Val Pro Val Gln
1 5 10 15
Gly Ser Lys
<210> 18
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 18
Gly Ile Pro Leu Glu Ser Thr Asp Gly Glu Arg
1 5 10
<210> 19
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 19
Ala Asp Glu Gly Ile Ser Phe Arg
1 5
<210> 20
<211> 26
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 20
Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln Ala Asn
1 5 10 15
His Pro Thr Ala Ala Val Val Thr Glu Lys
20 25
<210> 21
<211> 10
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 21
Ser Leu His Leu Glu Ala Ser Leu Asp Lys
1 5 10
<210> 22
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 22
Phe Gln Ala Glu Ile Pro Asp Arg
1 5
<210> 23
<211> 12
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 23
Glu Lys Pro Ala Asp Met Gln Asn Phe Gly Leu Arg
1 5 10
<210> 24
<211> 10
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 24
Thr Leu Leu Ala Asp Gln Gly Glu Ile Arg
1 5 10
<210> 25
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 25
Ile Glu Glu Asp Phe Pro His Ile Arg
1 5
<210> 26
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 26
Ile Met Glu Ala Pro Ile Pro Lys
1 5
<210> 27
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 27
Trp His Ile Asp Thr Ile Met Arg
1 5
<210> 28
<211> 7
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 28
Phe Pro Pro Tyr Tyr Met Arg
1 5
<210> 29
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 29
Gly Gln Glu Ser Glu Tyr Gly Asn Ile Thr Tyr Pro Pro Ala Val Arg
1 5 10 15
<210> 30
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 30
Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln His Asn Leu Arg
1 5 10
<210> 31
<211> 19
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 31
Glu Leu Ala Thr Asn Leu Asn Val Gly Thr Ser Ser Ser Ala Ala Ser
1 5 10 15
Ser Leu Lys
<210> 32
<211> 25
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 32
Thr Pro Val Val Gln Asn Ala Ala Ser Ile Val Gln Pro Ser Pro Ala
1 5 10 15
His Val Gly Gln Gln Gly Leu Ser Lys
20 25
<210> 33
<211> 30
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 33
Val Ser His Tyr Ile Ile Asn Ser Ser Gly Pro Arg Pro Pro Val Pro
1 5 10 15
Pro Ser Pro Ala Gln Pro Pro Pro Gly Val Ser Pro Ser Arg
20 25 30
<210> 34
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 34
Asn Leu Met Ile Asp Ile Gln Lys
1 5
<210> 35
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 35
Tyr Tyr Glu Ala Ala Asp Thr Val Thr Gln Phe Asp Asn Val Arg
1 5 10 15
<210> 36
<211> 10
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 36
Leu Val Gln Ala Phe Gln Phe Thr Asp Lys
1 5 10
<210> 37
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 37
Ile Pro Glu Leu Leu Ser Gly Gly Ser Val Asp Ser Glu Thr Arg
1 5 10 15
<210> 38
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 38
His Phe Glu Glu Leu Glu Thr Ile Met Asp Arg
1 5 10
<210> 39
<211> 18
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 39
Asp Ser Ser Thr Ser Pro Gly Asp Tyr Val Leu Ser Val Ser Glu Asn
1 5 10 15
Ser Arg
<210> 40
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 40
Ala Asn Phe Asp Ser Trp Ile Gly Leu His Arg
1 5 10
<210> 41
<211> 12
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 41
Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg
1 5 10
<210> 42
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 42
Leu Pro Leu Ala Thr Ile Val Lys
1 5
<210> 43
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 43
Leu Leu Val Glu Leu Val Gln Lys
1 5
<210> 44
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 44
Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala Asn Tyr Val Lys
1 5 10 15
<210> 45
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 45
Phe His Asp Leu Leu Ser Gln Leu Asp Asp Gln Tyr Ser Arg
1 5 10
<210> 46
<211> 18
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 46
Gln Gln Ala His Asp Leu Leu Ile Asn Lys Pro Asp Gly Thr Phe Leu
1 5 10 15
Leu Arg
<210> 47
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 47
Gln Gly Ile Ser Trp Ser Pro Glu Glu Ile Glu Asp Ala Arg
1 5 10
<210> 48
<211> 10
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 48
Ser Thr Asp Val Glu Val Phe Leu Pro Lys
1 5 10
<210> 49
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 49
Asn Trp Thr Glu Asp Ile Glu Gly Gly Ile Ser Ser Pro Val Lys
1 5 10 15
<210> 50
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 50
Ser Ile Met Ile Ser Gly Asn Val Leu Pro Asp Ala Thr Arg
1 5 10
<210> 51
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 51
Glu Asn Val Ser Asp Pro Ser Leu Thr Ile Thr Phe Gly Arg
1 5 10
<210> 52
<211> 7
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 52
Leu Phe Glu Leu Leu Glu Lys
1 5
<210> 53
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 53
Ile His Tyr Leu Asp Thr Thr Thr Leu Ile Glu Pro Val Ala Arg
1 5 10 15
<210> 54
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 54
Leu Gly Val Val Asp Val Phe Gln Glu Asp Lys
1 5 10
<210> 55
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 55
Asp Met Gly Val Tyr Thr Leu Asp Met Thr Asp Glu Asn Tyr Arg
1 5 10 15
<210> 56
<211> 19
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 56
Ala Ser Leu Pro Met Leu Ser Pro Thr Gly Ser Pro Gln Glu Val Glu
1 5 10 15
Val Gly Lys
<210> 57
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 57
Gln Ala Pro Glu Trp Thr Glu Glu Asp Leu Ser Gln Leu Thr Arg
1 5 10 15
<210> 58
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 58
Leu Ser Ala Met Pro Val Pro Phe Thr Pro Glu Leu Lys Pro Lys
1 5 10 15
<210> 59
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 59
Val Leu Ala Ile Asn Ile Leu Gly Arg
1 5
<210> 60
<211> 13
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 60
Ser Asp Asp Ser Val Ile Gln Leu Leu Asn Pro Asn Arg
1 5 10
<210> 61
<211> 23
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 61
Tyr Leu Glu Val Gln Tyr Lys Pro Gln Val His Ile Gln Met Thr Tyr
1 5 10 15
Pro Leu Gln Gly Leu Thr Arg
20
<210> 62
<211> 12
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 62
Phe Asp Asn Gln Asp Glu Leu Asn Phe Leu Met Arg
1 5 10
<210> 63
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 63
Ile Ala Pro Val His Ile Asp Thr Glu Ser Ile Ser Ala Leu Ile Lys
1 5 10 15
<210> 64
<211> 17
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 64
Gly Leu Val Glu Phe Gln Asp Val Ser Phe Ala Tyr Pro Asn Gln Pro
1 5 10 15
Lys
<210> 65
<211> 27
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 65
Leu Ser Gly Asp Leu Asn Ser Ile Gln Pro Ser Gly Ala Leu Ser Val
1 5 10 15
His Leu Ser Pro Pro Gln Thr Val Leu Ser Arg
20 25
<210> 66
<211> 18
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 66
Phe Leu Glu Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pro Met Glu
1 5 10 15
Ile Arg
<210> 67
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 67
Trp Ala Ile Leu Gly Leu Gly Val Arg
1 5
<210> 68
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 68
Gln Gly Gly Leu Gly Pro Met Asn Ile Pro Leu Ile Ser Asp Pro Lys
1 5 10 15
<210> 69
<211> 12
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 69
Glu Leu Ser Ala Val Thr Phe Pro Asp Ile Ile Arg
1 5 10
<210> 70
<211> 13
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 70
Tyr Asp Asp Val Leu Ile Asn Gly Leu Pro Asp Trp Arg
1 5 10
<210> 71
<211> 21
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 71
Phe Val Val Asn Phe Gln Asn Ser Phe Asn Gly Asn Asp Ile Ala Phe
1 5 10 15
His Phe Asn Pro Arg
20
<210> 72
<211> 12
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 72
Ser Leu Val Ala Leu Leu Val Glu Thr Gln Met Lys
1 5 10
<210> 73
<211> 32
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 73
Thr Pro Val Val Thr Gly Thr Gly Pro Asn Phe Ser Leu Gly Glu Leu
1 5 10 15
Gln Gly His Leu Ala Tyr Asp Leu Asn Pro Ala Ser Ala Gly Met Arg
20 25 30
<210> 74
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 74
Ala Thr Gln Leu Leu Glu Gly Leu Val Gln Glu Leu Gln Lys
1 5 10
<210> 75
<211> 33
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 75
Val Ser Gln Thr Pro Ile Ala Ala Gly Thr Gly Pro Asn Phe Ser Leu
1 5 10 15
Ser Asp Leu Glu Ser Ser Ser Tyr Tyr Ser Met Ser Pro Gly Ala Met
20 25 30
Arg
<210> 76
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 76
Leu Gly Asp Leu Asn Tyr Leu Ile Tyr Val Phe Pro Asp Arg Pro Lys
1 5 10 15
<210> 77
<211> 25
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 77
Ala Met Met Pro Gly Glu His Gly Ser Val Leu Ile Asp Ser Val Pro
1 5 10 15
Glu Val Pro Phe Pro Leu Ala Ser Lys
20 25
<210> 78
<211> 21
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 78
Asp Ile Gly Glu Gly Asn Leu Ser Thr Ala Ala Ala Ala Ala Leu Ala
1 5 10 15
Ala Ala Ala Val Lys
20
<210> 79
<211> 20
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 79
Leu Gly Phe Asp Thr Leu His Gly Leu Val Ser Thr Leu Ser Ala Gln
1 5 10 15
Pro Ser Leu Lys
20
<210> 80
<211> 21
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 80
Lys Pro Leu Leu Leu Ile Leu Asp Asp Ala Thr Ser Ala Leu Asp Ala
1 5 10 15
Gly Asn Gln Leu Arg
20
<210> 81
<211> 19
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 81
Leu Ser Ile Gln Asp Asn Asn Val Asp Leu Ile Leu Ala Thr Pro Pro
1 5 10 15
Phe Ser Arg
<210> 82
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 82
Met Leu Ser Leu Asn Val Phe Ser Val Gln Val Gln Ala Phe Lys
1 5 10 15
<210> 83
<211> 17
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 83
Ala Thr Leu Leu Asp Trp Val Ala Ser Glu Pro Leu Leu Ser Pro Gly
1 5 10 15
Arg
<210> 84
<211> 27
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 84
Thr Phe Gln Leu Gln Leu Leu Ser Pro Ser Ser Ser Val Val Pro Ala
1 5 10 15
Phe Asn Thr Gly Thr Ile Thr Gln Val Ile Lys
20 25
<210> 85
<211> 21
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 85
Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr Gln Gln Gln Gly
1 5 10 15
Ile Leu Gln Asp Arg
20
<210> 86
<211> 24
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 86
Thr Ile Ser Pro Glu His Val Ile Gln Ala Leu Glu Ser Leu Gly Phe
1 5 10 15
Gly Ser Tyr Ile Ser Glu Val Lys
20
<210> 87
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 87
Val Gly Leu Glu Ile Pro Gly Glu Met Trp Leu Ser Trp Val Pro Arg
1 5 10 15
<210> 88
<211> 21
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 88
Thr Ala Gln Ala Ile Glu Pro Tyr Ile Thr Asn Phe Phe Asn Gln Val
1 5 10 15
Leu Met Leu Gly Lys
20
<210> 89
<211> 19
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 89
Thr Leu Ala Gly Leu Val Val Gln Leu Leu Gln Phe Gln Glu Asp Ala
1 5 10 15
Phe Gly Lys
<210> 90
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 90
Ala Pro Ile Arg Pro Asp Ile Val Asn Phe Val His Thr Asn Leu Arg
1 5 10 15
<210> 91
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 91
Ile Glu Glu Val Pro Glu Leu Pro Leu Val Val Glu Asp Lys
1 5 10
<210> 92
<211> 12
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 92
Asn Ile Pro Gly Ile Thr Leu Leu Asn Val Ser Lys
1 5 10
<210> 93
<211> 13
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 93
Ile Val Gln Leu Leu Ala Gly Val Ala Asp Gln Thr Lys
1 5 10
<210> 94
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 94
Ile Tyr Ser Thr Leu Ala Gly Thr Arg
1 5
<210> 95
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 95
Leu Thr Val Asn Asp Phe Val Arg
1 5
<210> 96
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 96
Asp Leu Glu Glu Phe Phe Ser Thr Val Gly Lys
1 5 10
<210> 97
<211> 23
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 97
Gly Ile Ala Tyr Val Glu Phe Val Asp Val Ser Ser Val Pro Leu Ala
1 5 10 15
Ile Gly Leu Thr Gly Gln Arg
20
<210> 98
<211> 17
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 98
Thr Asp Ala Ser Ser Ala Ser Ser Phe Leu Asp Ser Asp Glu Leu Glu
1 5 10 15
Arg
<210> 99
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 99
Glu Ala Ala Leu Gly Ala Gly Phe Ser Asp Lys
1 5 10
<210> 100
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 100
Leu Glu Gln Asp Glu Tyr Ala Leu Arg
1 5
<210> 101
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 101
Asn Val Val Val Val Asp Gly Val Arg
1 5
Claims (13)
1. A method for detecting a protein in a complex sample, the method comprising:
performing chromatographic separation and mass spectrum scanning on the complex sample to acquire mass spectrum data, wherein in the process of acquiring the mass spectrum data by mass spectrum scanning, scanning acquisition of a PRM mode is inserted in the process of DDA mode scanning acquisition, the scanning acquisition of the PRM mode is arranged in different time windows in the DDA mode scanning acquisition process, the DDA mode is extruded by the PRM mode in the same time window, and the time of the data acquired by the PRM mode occupies the time of the data acquired by the DDA mode;
performing qualitative and quantitative analysis on the protein in the mass spectrum data to obtain the expression information of the full-spectrum protein of the complex sample;
and in the complex sample, scanning and acquiring the PRM mode in a time window corresponding to the target peptide segment of the target protein.
2. The method of claim 1, wherein the DDA mode scan and the scan acquisition of the PRM mode are performed using a quadrupole mass spectrometer or a triple spectrometer.
3. The method of claim 1, wherein the step of quantitatively analyzing the protein in the mass spectral data comprises:
carrying out quantitative analysis on the mass spectrum data acquired by the DDA mode according to the peak area of the primary mass spectrum;
and carrying out quantitative analysis according to the addition of the first three high secondary fragment ions in the targeting peptide fragment by using the mass spectrum data acquired by the PRM mode.
4. The method of any one of claims 1 to 3, wherein the complex sample is chromatographically separated using a chromatography column, wherein the chromatography column comprises:
the outer diameter of the chromatographic column tube is 355-365 mu m, and the inner diameter of the chromatographic column tube is 100-200 mu m;
the packing is filled in the chromatographic column tube, and the packing is C18 packing; and
and the chromatographic column tip is integrally designed with the chromatographic column tube.
5. The method of claim 4, wherein the length of the chromatography column tube is 150-300 mm.
6. The method of claim 4, wherein the inner diameter of the chromatographic column tip is 2-8 μm.
7. The method of claim 6, wherein the inner diameter of the chromatographic column tip is 3 to 6 μm.
8. The method according to claim 6, wherein the filler has a particle size of 1.5 to 3 μm.
9. The method of claim 8, wherein the filler has a particle size of 1.8 to 2.5 μm.
10. The method according to claim 4, wherein in the step of chromatographic separation, a chromatographic separation gradient of 75-150 min is adopted for separation.
11. The method of any one of claims 1 to 3, wherein the complex sample is a tissue sample, a cell sample, or a body fluid sample.
12. The method according to any one of claims 1 to 3, wherein the complex sample is a plant sample, an animal sample or a microbial sample.
13. The method of claim 1, wherein the target protein is one or more proteins in the mass spectral historical data acquired based on the DDA mode.
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