CN113588833A - Reference product for monitoring proteolysis efficiency and false cutting rate in preparation of proteome sample and application thereof - Google Patents
Reference product for monitoring proteolysis efficiency and false cutting rate in preparation of proteome sample and application thereof Download PDFInfo
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- CN113588833A CN113588833A CN202110890732.2A CN202110890732A CN113588833A CN 113588833 A CN113588833 A CN 113588833A CN 202110890732 A CN202110890732 A CN 202110890732A CN 113588833 A CN113588833 A CN 113588833A
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- 108010026552 Proteome Proteins 0.000 title claims abstract description 27
- 238000012544 monitoring process Methods 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 230000017854 proteolysis Effects 0.000 title claims abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 85
- 102000007079 Peptide Fragments Human genes 0.000 claims abstract description 76
- 108010033276 Peptide Fragments Proteins 0.000 claims abstract description 76
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 3
- 238000003776 cleavage reaction Methods 0.000 claims description 20
- 230000007017 scission Effects 0.000 claims description 19
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 239000007791 liquid phase Substances 0.000 claims description 7
- 239000013558 reference substance Substances 0.000 claims description 7
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 4
- 238000001819 mass spectrum Methods 0.000 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 230000000155 isotopic effect Effects 0.000 claims description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 238000004364 calculation method Methods 0.000 claims description 2
- 238000013075 data extraction Methods 0.000 claims description 2
- 238000001948 isotopic labelling Methods 0.000 claims description 2
- 238000004949 mass spectrometry Methods 0.000 claims description 2
- 230000002797 proteolythic effect Effects 0.000 claims 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000005932 reductive alkylation reaction Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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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
-
- 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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- 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
-
- 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
- G01N2030/8818—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 amino acids
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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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
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Abstract
The invention discloses a reference product for monitoring the proteolysis efficiency and the false cutting rate in the preparation of a proteome sample and application thereof, wherein an exogenous peptide fragment group standard product is designed according to the required conditions and is connected in series with a peptide fragment to form a long peptide standard product, the concentrations of the two are consistent, and the isotope labels are different; respectively adding the long peptide standard substance and the peptide fragment group standard substance before and after the enzymolysis step in the proteome extraction process, and concomitantly completing the subsequent process; after the protein group is extracted, a peptide fragment sample is obtained, liquid quality detection is used, and the intensity proportion of corresponding peptide fragments in a peptide fragment group standard product and a long peptide standard product is compared, so that the enzymolysis efficiency and the truncation ratio can be calculated. The reference product can effectively monitor the enzymolysis efficiency and the miscut efficiency of the protein.
Description
Technical Field
The invention belongs to the technical field of proteomics, and particularly relates to a reference substance for monitoring proteolysis efficiency and false cutting rate in preparation of a proteome sample and application thereof.
Background
With the development of instruments and equipment and detection technology, the proteome macro data detection method develops very rapidly and relates to various scientific researches, clinical medical diagnosis, early screening, prognosis monitoring and the like.
Correspondingly, various proteome sample pretreatment methods appear in the market, including the steps of extraction, purification, enzymolysis and the like, but no proper monitoring standard exists for the extraction efficiency and accuracy of different methods.
In the current proteome extraction method, the enzymolysis efficiency greatly affects the detection efficiency, and the enzymolysis efficiency is low, so that the mass spectrum response intensity of the finally detected peptide fragments and the number of the detected peptide fragments can be reduced, the quantitative data of the protein can be affected, and the deviated proteome quantitative data can be obtained. The current enzymolysis efficiency is only that the types of uncut peptide fragments identified in the judgment result account for the total number of the identified types of the peptide fragments, and the proportion of the successfully enzymolyzed protein, namely the enzymolysis efficiency cannot be actually judged. Meanwhile, for the commonly used trypsin, no method is available for monitoring the efficiency of the mis-cutting, namely, continuous proline is in front of arginine/lysine at the enzyme cutting site, and the mis-cutting cannot be cut, but the probability exists.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems, the invention provides a reference substance for monitoring the proteolysis efficiency and the false cutting rate in the preparation of a proteome sample and application thereof.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
a reference product for monitoring proteolysis efficiency and false cutting rate in preparation of proteome sample comprises a group of exogenous peptide fragment group standard products and a long peptide standard product;
the design of the exogenous peptide fragment group standard product is as follows:
1) each peptide segment is ended by arginine (R) or lysine (K), no special indication is made, and no R or K exists at other positions of the peptide segment;
2) the length of at least one peptide fragment A1 is slightly less than the maximum detection length of the applicable liquid phase mass spectrometer;
3) at least one of the peptides A2 comprises a proline-arginine structure (PR) which is at least 7 amino acids long before or after the structure;
4) at least one of the peptides A3 comprises a proline-lysine structure (PK) which is at least 7 amino acids long before or after the structure;
5) at least 3 basic peptide fragments B are provided, and the polarities of the three peptide fragments 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 segment in the exogenous peptide segment group standard product is different from the corresponding peptide segment in the long peptide standard product in isotope labeling; the molar ratio of the peptide segments in the exogenous peptide segment group standard is the same as that of the corresponding peptide segments in the long peptide standard.
Preferably, the length of the peptide fragment A1 is as follows: the length of the detection is 5-7 amino acids less than the maximum detection length of an applicable mass spectrometer, and the length of the rest peptide fragments is 7-14 amino acids.
Preferably, the polarity of the 3 basic peptide fragments B is: can be eluted from the liquid chromatographic column by 5-30%, 30-60% and 60-95% acetonitrile water solution respectively.
Preferably, the isotopic labels differ by: each peptide segment in the exogenous peptide segment group standard product is marked by an isotope, and the corresponding peptide segment in the long peptide standard product is not marked by the isotope; each peptide segment in the exogenous peptide segment group standard sample is not marked by an isotope, and the corresponding peptide segments in the long peptide standard sample are marked by isotopes; or each peptide segment in the exogenous peptide segment group standard product and the long peptide standard product is marked by an isotope, but the isotopes used for marking are different. Preferably the label K or R.
Preferably, the molar ratio between the peptide fragments in the exogenous peptide fragment group standard and the molar ratio between the corresponding peptide fragments in the long peptide standard are the same, preferably both are 1:1:1 …:1, and can be other integer ratios.
The invention also provides application of the reference substance in monitoring the proteolysis efficiency and the false cutting rate in the preparation of the proteome sample.
During specific application, the long peptide standard is added into the proteome experiment process, enzymolysis is carried out together with a sample, and then the equivalent peptide fragment group standard is added into the post-enzymolysis step for monitoring the enzymolysis efficiency and the miscut rate.
The invention finally provides a method for monitoring the proteolysis efficiency and the false cleavage rate in the preparation of a proteome sample, which comprises the following steps:
taking the reference product, adding the long peptide standard product into the proteome experiment process, carrying out enzymolysis together with the sample, and adding the equivalent peptide fragment group standard product into the post-enzymolysis step for monitoring the enzymolysis efficiency and the miscut rate; after proteome extraction is finished, performing proteome detection by using liquid phase mass spectrometry, performing data extraction analysis by using corresponding software, and quantifying each piece of peptide fragment information of the peptide fragment group, wherein a calculation formula is as follows:
the enzyme cutting efficiency is the median (peptide fragment B in the long peptide standard/peptide fragment B in the peptide fragment group standard);
PR cleavage error rate (a 2 in the long peptide sample/a 2 in the peptide fragment group sample)/cleavage efficiency;
the PK cleavage error rate (A3 in long peptide sample/A3 in peptide fragment sample)/cleavage efficiency.
Preferably, the equivalent criteria are: the peptide fragment group standard substance is quantified by the peptide fragment with the minimum molar weight in each peptide fragment, and the long peptide standard substance is quantified by the molar weight of the long peptide body.
Has the advantages that: compared with the prior art, the invention selects reasonable peptide segment combination standard products, including 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 long peptide standard products. The long peptide standard and the peptide fragment standard are added into the proteome extraction enzymolysis process, so that the enzymolysis efficiency and the miscut efficiency of the protein can be monitored.
Detailed Description
The following is a general description of the embodiments of the present invention, which are the most preferred embodiments of the present invention, but the present invention is not limited to the following examples.
1. Design and Synthesis of peptide fragments
1) According to the requirements and the preferred requirements of the reference substances in the above summary of the invention, a peptide fragment group is designed and synthesized, and isotopic labels are used on K and R, as shown in Table 1;
TABLE 1 peptide fragment sequences
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 product;
2. monitoring protein loss during extraction using protein standards
1) Taking a cell sample as an example, collecting cells with a volume of about 50uL, and adding a lysis solution;
2) incubating on ice for 15min, and ultrasonically crushing for 10 min;
3) carrying out reductive alkylation on the sample;
4) taking out 100ug of protein, adding sp3 magnetic beads for adsorption, and cleaning the magnetic beads;
5) adding a 19uL trypsin enzymolysis system into 1uL long peptide standard (100f/10ug, quantitative by the molar weight of the long peptide body) in the adsorption magnetic beads, and carrying out enzymolysis at 37 ℃ overnight;
6) adding 1uL peptide fragment group standard (100f/10ug, quantitative determination is carried out by using the peptide fragment with the minimum molar weight in each peptide fragment), adding 400uL acetonitrile, carrying out peptide fragment adsorption, and washing by using the acetonitrile;
7) adding 50uL eluent to elute the peptide segment;
8) injecting 10uL of sample, and detecting the sample by using a liquid phase mass spectrum through a DIA method;
9) protein identification and quantification are carried out by using Spectronaut software;
10) extracting the peptide fragment corresponding to the standard, as shown in table 3, the protease cleavage efficiency is medium (peptide fragment B in long peptide standard/peptide fragment B in peptide fragment group standard), PR cleavage error rate is (a 2 in long peptide standard/a 2 in peptide fragment group standard)/enzyme cleavage efficiency, PK cleavage error rate is (A3 in long peptide standard/A3 in peptide fragment group standard)/enzyme cleavage efficiency
TABLE 3 Ionic Strength and recovery of standard peptides in samples
3. And (4) conclusion:
1) as can be seen from table 3, the median recovery rate of the B1-B3 peptide fragments in the long peptide standard was 95.5%, which indicates that the recovery rate of the whole extraction and purification of the sample was 95.5%;
2) the recovery rates of A2 and A3 are 93.8 percent and 92.2 percent respectively, the false cutting rates are 98.2 percent and 96.5 percent respectively, the difference is within 2 percent and 4 percent respectively, 2 percent is an acceptable error range, the PR false cutting influence is not large, the 4 percent error is slightly large, but still acceptable, and proper attention is needed;
3) the recovery rate of A1 was 94.9%, and the difference was not large, indicating that the digestion efficiency of the long peptide fragment was good.
Claims (9)
1. A reference product for monitoring the proteolysis efficiency and the false cutting rate in the preparation of a proteome sample is characterized by comprising a group of exogenous peptide fragment group standard products and a long peptide standard product;
the design of the exogenous peptide fragment group standard product is as follows:
1) each peptide segment is ended by arginine (R) or lysine (K), no special indication is made, and no R or K exists at other positions of the peptide segment;
2) the length of at least one peptide fragment A1 is slightly less than the maximum detection length of the applicable liquid phase mass spectrometer;
3) at least one of the peptides A2 comprises a proline-arginine structure (PR) which is at least 7 amino acids long before or after the structure;
4) at least one of the peptides A3 comprises a proline-lysine structure (PK) which is at least 7 amino acids long before or after the structure;
5) at least 3 basic peptide fragments B are provided, and the polarities of the three peptide fragments 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 segment in the exogenous peptide segment group standard product is different from the corresponding peptide segment in the long peptide standard product in isotope labeling; the molar ratio of the peptide segments in the exogenous peptide segment group standard is the same as that of the corresponding peptide segments in the long peptide standard.
2. The reference substance for monitoring the proteolytic efficiency and the false cleavage rate in the preparation of a proteome sample according to claim 1, wherein the length of the peptide fragment A1 is: the length of the detection is 5-7 amino acids less than the maximum detection length of an applicable mass spectrometer, and the length of the rest peptide fragments is 7-14 amino acids.
3. The reference product for monitoring the proteolytic efficiency and the false cleavage rate in the preparation of a proteome sample according to claim 1, wherein the polarity of the 3 basic peptide fragments B is: can be eluted from the liquid chromatographic column by 5-30%, 30-60% and 60-95% acetonitrile water solution respectively.
4. The reference product for monitoring the proteolytic efficiency and the false cleavage rate in the preparation of a proteomic sample, according to claim 1, wherein the isotopic labels are different and comprise: each peptide segment in the exogenous peptide segment group standard product is marked by an isotope, and the corresponding peptide segment in the long peptide standard product is not marked by the isotope; each peptide segment in the exogenous peptide segment group standard sample is not marked by an isotope, and the corresponding peptide segments in the long peptide standard sample are marked by isotopes; or each peptide segment in the exogenous peptide segment group standard product and the long peptide standard product is marked by an isotope, but the isotopes used for marking are different.
5. The reference product for monitoring the proteolytic efficiency and the false cleavage rate in the preparation of a proteome sample according to claim 1, wherein the molar ratio between each peptide fragment in the exogenous peptide fragment group standard product and the molar ratio between the corresponding peptide fragments in the long peptide standard product are the same, preferably 1:1: 1:1 …: 1.
6. Use of a reference substance according to any one of claims 1 to 5 for monitoring the efficiency of proteolysis and the rate of mis-cleavage in the preparation of a proteomic sample.
7. The use of claim 6, wherein the long peptide is added to the proteome experiment process, and is subjected to enzymolysis together with the sample, and then the equivalent peptide fragment group standard is added to the post-enzymolysis step for monitoring the enzymolysis efficiency and the false cutting rate.
8. A method for monitoring the proteolysis efficiency and the false cleavage rate in the preparation of a proteome sample is characterized by comprising the following steps:
taking the reference substance of any one of claims 1-5, adding a long peptide standard substance into a proteome experiment process, carrying out enzymolysis together with a sample, and adding an equivalent peptide fragment group standard substance into a post-enzymolysis step for monitoring enzymolysis efficiency and miscut rate; after proteome extraction is finished, performing proteome detection by using liquid phase mass spectrometry, performing data extraction analysis by using corresponding software, and quantifying each piece of peptide fragment information of the peptide fragment group, wherein a calculation formula is as follows:
the enzyme cutting efficiency is the median (peptide fragment B in the long peptide standard/peptide fragment B in the peptide fragment group standard);
PR cleavage error rate (a 2 in the long peptide sample/a 2 in the peptide fragment group sample)/cleavage efficiency;
the PK cleavage error rate (A3 in long peptide sample/A3 in peptide fragment sample)/cleavage efficiency.
9. The method of claim 8, wherein the equivalence criterion is: the peptide fragment group standard substance is quantified by the peptide fragment with the minimum molar weight in each peptide fragment, and the long peptide standard substance is quantified by the molar weight of the long peptide body.
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Citations (5)
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| FR2832508A1 (en) * | 2001-11-21 | 2003-05-23 | Bio Merieux | Preparing selective capture and detection phases, useful in immunological assay of biomolecules, using binding molecules modified by polyhistidine and polylysine tags |
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| CN107102149A (en) * | 2017-05-03 | 2017-08-29 | 杭州帕匹德科技有限公司 | A kind of Protein in Food quantitatively detects the screening technique with feature peptide fragment |
| CN107384998A (en) * | 2016-05-16 | 2017-11-24 | 中国科学院大连化学物理研究所 | A kind of protein C based on carboxypeptidase and strong cation exchange chromatography-end enrichment method |
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2021
- 2021-08-04 CN CN202110890732.2A patent/CN113588833B/en active Active
Patent Citations (5)
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| FR2832508A1 (en) * | 2001-11-21 | 2003-05-23 | Bio Merieux | Preparing selective capture and detection phases, useful in immunological assay of biomolecules, using binding molecules modified by polyhistidine and polylysine tags |
| US20120021446A1 (en) * | 2009-02-23 | 2012-01-26 | Tohoku University | Evaluation Peptide For Use In Quantification Of Protein Using Mass Spectrometer, Artificial Standard Protein, And Method For Quantifying Protein |
| CN104569134A (en) * | 2015-01-06 | 2015-04-29 | 中国计量科学研究院 | Method for accurately testing digestion efficiency of proteins in matrix |
| CN107384998A (en) * | 2016-05-16 | 2017-11-24 | 中国科学院大连化学物理研究所 | A kind of protein C based on carboxypeptidase and strong cation exchange chromatography-end enrichment method |
| CN107102149A (en) * | 2017-05-03 | 2017-08-29 | 杭州帕匹德科技有限公司 | A kind of Protein in Food quantitatively detects the screening technique with feature peptide fragment |
Non-Patent Citations (3)
| Title |
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| DOROTHÉE LEBERT等: "DIGESTIF: A Universal Quality Standard for the Control of Bottom-Up Proteomics Experiments", 《J. PROTEOME RES.》 * |
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