CN113588833B - Reference for monitoring proteolysis efficiency and miscut rate in proteome sample preparation and application thereof - Google Patents
Reference for monitoring proteolysis efficiency and miscut rate in proteome sample preparation and application thereof Download PDFInfo
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- CN113588833B CN113588833B CN202110890732.2A CN202110890732A CN113588833B CN 113588833 B CN113588833 B CN 113588833B CN 202110890732 A CN202110890732 A CN 202110890732A CN 113588833 B CN113588833 B CN 113588833B
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- 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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- 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
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Abstract
The invention discloses a reference for monitoring proteolytic efficiency and miscut rate in proteome sample preparation and application thereof, wherein the invention designs exogenous peptide fragment group standard according to the required conditions, and connects peptide fragments in series to form a peptide standard, the concentration of the two peptide standard is consistent, and isotope labels are different; respectively adding the long peptide standard and the peptide fragment group standard before and after the enzymolysis step in the proteome extraction process, and completing the subsequent process; and (3) extracting the protein group, obtaining a peptide fragment sample, and comparing the intensity ratio of the corresponding peptide fragments in the peptide fragment group standard and the long peptide standard by using liquid quality detection, so that the enzymolysis efficiency and the miscut rate can be calculated. The reference product can effectively monitor the enzymolysis efficiency and miscut efficiency of the protein.
Description
Technical Field
The invention belongs to the technical field of proteomics, and particularly relates to a reference for monitoring proteolysis efficiency and miscut rate in proteome sample preparation and application thereof.
Background
With the development of instruments and equipment and detection technology, the macroscopic big data detection method of the proteome is very rapid in development, and relates to various scientific researches, clinical medical diagnosis, early screening, prognosis monitoring and the like.
Accordingly, various methods for pretreatment of proteome samples are presented in the market, including steps of extraction, purification, enzymolysis and the like, but the extraction efficiency and accuracy of different methods do not have proper monitoring standards.
In the current proteome extraction method, the enzymolysis efficiency greatly influences the detection efficiency, and if the enzymolysis efficiency is low, the mass spectrum response intensity of the final detected peptide fragments and the quantity of the detected peptide fragments can be reduced, and the protein quantitative data is influenced, so that the proteome quantitative data with deviation can be obtained. The current enzymolysis efficiency is only that the types of the uncut peptide fragments identified in the evaluation result account for the total number of the identified peptide fragment fragments, and the proportion of the successfully hydrolyzed protein, namely the enzymolysis efficiency, cannot be actually evaluated. Meanwhile, for the common trypsin, no method is used for monitoring the miscut efficiency at present, namely, continuous proline exists before arginine/lysine at the enzyme cleavage site, the enzyme cannot be cleaved, but the miscut probability exists.
Disclosure of Invention
The invention aims to: in order to solve the technical problems, the invention provides a reference for monitoring the proteolytic efficiency and the miscut rate in the preparation of proteome samples and application thereof.
The technical scheme is as follows: in order to achieve the aim of the invention, the invention adopts the following technical scheme:
a reference for monitoring proteolytic efficiency and miscut rate in proteome sample preparation comprises a group of exogenous peptide fragment group standard and a long peptide standard;
the design of the exogenous peptide fragment group standard is as follows:
1) Each peptide is terminated by arginine (R) or lysine (K), and no special description exists at other positions of the peptide;
2) The length of at least one peptide fragment A1 is slightly smaller than the maximum detection length of a liquid phase mass spectrometer;
3) At least one peptide fragment A2 comprises a proline-arginine structure (PR) and the structure is preceded or followed by a length of at least 7 amino acids;
4) At least one peptide fragment A3 comprises a proline-lysine structure (PK) and the structure is preceded or followed by a length of at least 7 amino acids;
5) At least 3 basic peptide segments B are provided, and the polarities of the three peptide segments can be evenly distributed in the detection range of the liquid phase mass spectrum;
the long peptide standard is formed by connecting all peptide fragments in the exogenous peptide fragment group standard end to end;
each peptide in the exogenous peptide fragment group standard is different in isotope labeling from the corresponding peptide fragment in the long peptide standard; the molar ratio between peptide fragments in the exogenous peptide fragment group standard is the same as the molar ratio between corresponding peptide fragments in the long peptide standard.
Preferably, the length of the peptide fragment A1 is: the maximum detection length of the mass spectrometer is 5-7 amino acids less than that of a suitable mass spectrometer, and the length of the rest peptide fragment is 7-14 amino acids.
Preferably, the polarities of the 3 basic peptide fragments B are divided into: can be eluted from the liquid chromatographic column by 5-30%,30-60%,60-95% acetonitrile water solution respectively.
Preferably, the isotopic labeling differences include: each peptide in the exogenous peptide group standard has isotope labeling, and the corresponding peptide in the long peptide is not isotope labeled; each peptide in the exogenous peptide group standard is not isotopically labeled, and the corresponding peptide in the long peptide is isotopically labeled; or the peptide fragments in the exogenous peptide fragment group standard and the long peptide standard are respectively labeled by isotopes, but the isotopes used for labeling are different. Preferably the label K or R.
Preferably, the molar ratio between the peptide fragments in the exogenous peptide fragment group standard is the same as the molar ratio between the corresponding peptide fragments in the long peptide standard, preferably 1:1:1 …:1, and can be other integer ratios.
The invention also provides application of the reference in monitoring proteolytic efficiency and miscut rate in preparation of proteome samples.
In specific application, the long peptide standard is added into the proteome experiment process, and is subjected to enzymolysis together with a sample, and then the equivalent peptide fragment standard is added into the step after enzymolysis, so that the enzymolysis efficiency and the miscut rate are monitored.
The invention finally provides a method for monitoring the proteolytic efficiency and the miscut rate in the preparation of a proteome sample, which comprises the following steps:
adding the long peptide standard into the proteome experiment process, carrying out enzymolysis together with the sample, and then adding the equivalent peptide standard into the step after enzymolysis for monitoring the enzymolysis efficiency and the miscut rate; after the proteome extraction is completed, the proteome detection is carried out by using liquid phase mass spectrum, the data extraction analysis is carried out by using corresponding software, and each piece of peptide fragment information of the peptide fragment group is quantified, wherein the calculation formula is as follows:
cleavage efficiency = median (peptide fragment B in long peptide standard/peptide fragment B in peptide fragment group standard);
PR miscut rate= (A2 in long peptide standard/A2 in peptide group standard)/enzyme digestion efficiency;
PK miscut rate= (A3 in long peptide standard/A3 in peptide group standard)/cleavage efficiency.
Preferably, the criteria for the equivalent amount are: the peptide fragment group standard is quantified by the peptide fragment with the smallest molar amount in each peptide fragment, and the long peptide standard is quantified by the molar amount of the long peptide body.
The beneficial effects are that: compared with the prior art, the invention selects reasonable peptide segment combination standard products, comprising peptide segments distributed in different polarities, peptide segments containing continuous PK and PR and ultra-long peptide segments, and then connects the short peptides into a long peptide standard product. The long peptide standard and the peptide fragment standard are added into the proteome extraction enzymolysis process, so that the enzymolysis efficiency and miscut efficiency of the protein can be monitored.
Detailed Description
The following description of the present invention will be given in terms of the best mode for carrying out the invention, but the present invention is not limited to the following examples.
1. Designing and synthesizing groups of peptide fragments
1) According to the requirements and the preferred requirements of the reference in the above summary, designing a peptide fragment group and synthesizing, and using isotope labels on K and R as shown in table 1;
TABLE 1 peptide fragment sequence
2) The sequences are connected end to end according to the sequence A1-B1-A2-B2-A3-B3 to synthesize a non-heavy standard long peptide standard;
2. monitoring protein loss during extraction using protein markers
1) Taking a cell sample as an example, a volume of about 50uL of cells is collected and lysates are added;
2) Incubating on ice for 15min, and performing ultrasonic crushing for 10min;
3) Carrying out sample reductive alkylation;
4) Taking out 100ug of protein, adding sp3 magnetic beads for adsorption, and cleaning the magnetic beads;
5) 1uL of long peptide standard (100 f/10ug, which is quantified by the molar quantity of the long peptide body) is added into the adsorption magnetic beads, and a 19uL trypsin enzymolysis system is added for enzymolysis at 37 ℃ overnight;
6) Adding 1uL of peptide fragment group standard (100 f/10ug, quantifying the peptide fragment with the smallest molar amount in each peptide fragment), adding 400uL of acetonitrile, adsorbing the peptide fragment, and cleaning by using acetonitrile;
7) Adding 50uL of eluent to elute the peptide fragment;
8) Sample injection is carried out by 10uL, and the liquid phase mass spectrum is used for detecting the sample by a DIA method;
9) Protein identification and quantification using spectrobaut software;
10 The peptide fragment corresponding to the standard was extracted, and as shown in table 3, the protease cleavage efficiency=median (peptide fragment B in long peptide standard/peptide fragment B in peptide fragment group standard), PR miscut rate= (A2 in long peptide standard/A2 in peptide fragment group standard)/enzyme cleavage efficiency, PK miscut rate= (A3 in long peptide standard/A3 in peptide fragment group standard)/enzyme cleavage efficiency
TABLE 3 ion intensity and recovery of standard peptide fragments in samples
3. Conclusion:
1) As can be seen from Table 3, the recovery ratio of the B1-B3 peptide fragment in the long peptide standard is 95.5%, which can indicate that the overall extraction and purification recovery ratio of the sample is 95.5%;
2) A2 and A3 recovery rates are 93.8% and 92.2% respectively, the miscut rate is 98.2% and 96.5% respectively, the difference is within 2% and 4% respectively, 2% is an acceptable error range, which indicates that the effect of PR miscut is not large, 4% error is slightly large, but still acceptable, and proper attention is needed;
3) A1 recovery rate is 94.9%, the difference is not large, and the digestion efficiency of the long peptide fragment is good.
Claims (9)
1. A reference for monitoring proteolytic efficiency and miscut rate in proteome sample preparation is characterized by comprising a group of exogenous peptide fragment group standard substances and a long peptide standard substance;
the design of the exogenous peptide fragment group standard is as follows:
1) Each peptide is terminated by arginine R or lysine K, and R or K is not present at other positions of the peptide;
2) The length of at least one peptide fragment A1 is slightly smaller than the maximum detection length of a liquid phase mass spectrometer;
3) At least one peptide fragment A2 comprises a proline-arginine structure PR, and the structure is preceded or followed by a length of at least 7 amino acids;
4) At least one peptide fragment A3 comprises the proline-lysine structure PK, and the structure is preceded or followed by a length of at least 7 amino acids;
5) At least 3 basic peptide segments B are provided, and the polarities of the three peptide segments can be evenly distributed in the detection range of the liquid phase mass spectrum;
the long peptide standard is formed by connecting all peptide fragments in the exogenous peptide fragment group standard end to end;
each peptide in the exogenous peptide fragment group standard is different in isotope labeling from the corresponding peptide fragment in the long peptide standard; the molar ratio between peptide fragments in the exogenous peptide fragment group standard is the same as the molar ratio between corresponding peptide fragments in the long peptide standard.
2. The reference for monitoring proteolytic efficiency and miscut rate in preparation of proteomic samples according to claim 1, wherein the length of the peptide fragment A1 is: the maximum detection length of the mass spectrometer is 5-7 amino acids less than that of a suitable mass spectrometer, and the length of the rest peptide fragment is 7-14 amino acids.
3. The reference for monitoring proteolytic efficiency and miscut rate in preparation of proteomic samples according to claim 1, wherein the polarities of the 3 basic peptide fragments B are divided into: can be eluted from the liquid chromatographic column by 5-30%,30-60%,60-95% acetonitrile water solution respectively.
4. The reference for monitoring proteolytic efficiency and miscut rate in the preparation of proteomic samples according to claim 1, wherein the isotopic labeling differences comprise: each peptide in the exogenous peptide group standard has isotope labeling, and the corresponding peptide in the long peptide is not isotope labeled; each peptide in the exogenous peptide group standard is not isotopically labeled, and the corresponding peptide in the long peptide is isotopically labeled; or the peptide fragments in the exogenous peptide fragment group standard and the long peptide standard are respectively labeled by isotopes, but the isotopes used for labeling are different.
5. The reference for monitoring proteolytic efficiency and miscut rate in preparation of proteomic samples according to claim 1, wherein the molar ratio between the peptide fragments in the exogenous peptide fragment group standard is the same as the molar ratio between the corresponding peptide fragments in the long peptide standard.
6. Use of a reference according to any one of claims 1 to 5 for monitoring proteolytic efficiency and miscut rate in the preparation of a proteomic sample.
7. The use according to claim 6, wherein the long peptide standard is added to the proteome test procedure for enzymolysis together with the sample, and the equivalent peptide standard is added to the post-enzymolysis step for monitoring the enzymolysis efficiency and miscut rate.
8. A method for monitoring proteolytic efficiency and miscut rate in the preparation of a proteome sample, comprising the steps of:
taking the reference substance as in any one of claims 1-5, adding a long peptide standard substance into the proteome experiment process, carrying out enzymolysis together with a sample, and then adding an equivalent peptide fragment standard substance into the enzymolysis step for monitoring the enzymolysis efficiency and the miscut rate; after the proteome extraction is completed, the proteome detection is carried out by using liquid phase mass spectrum, the data extraction analysis is carried out by using corresponding software, and each piece of peptide fragment information of the peptide fragment group is quantified, wherein the calculation formula is as follows:
cleavage efficiency = median (peptide fragment B in long peptide standard/peptide fragment B in peptide fragment group standard);
PR miscut rate= (A2 in long peptide standard/A2 in peptide group standard)/enzyme digestion efficiency;
PK miscut rate= (A3 in long peptide standard/A3 in peptide group standard)/cleavage efficiency.
9. The method of claim 8, wherein the criteria for the same amount are: the peptide fragment group standard is quantified by the peptide fragment with the smallest molar amount in each peptide fragment, and the long peptide standard is quantified by the molar amount of the long peptide body.
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| JP5317336B2 (en) * | 2009-02-23 | 2013-10-16 | 国立大学法人東北大学 | Peptide for evaluation, artificial standard protein, and protein quantification method for protein quantification using mass spectrometer |
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| CN107102149B (en) * | 2017-05-03 | 2019-03-29 | 杭州帕匹德科技有限公司 | A kind of screening technique of Protein in Food quantitative detection feature peptide fragment |
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