WO2019013256A1 - Procédé d'évaluation de la qualité d'un échantillon biologique et marqueur associé - Google Patents

Procédé d'évaluation de la qualité d'un échantillon biologique et marqueur associé Download PDF

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WO2019013256A1
WO2019013256A1 PCT/JP2018/026202 JP2018026202W WO2019013256A1 WO 2019013256 A1 WO2019013256 A1 WO 2019013256A1 JP 2018026202 W JP2018026202 W JP 2018026202W WO 2019013256 A1 WO2019013256 A1 WO 2019013256A1
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peptide
stable
peptides
quality
variable
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PCT/JP2018/026202
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English (en)
Japanese (ja)
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毅 朝長
白水　崇
義男 小寺
七里　眞義
晃一郎 湯地
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国立研究開発法人医薬基盤・健康・栄養研究所
学校法人北里研究所
国立大学法人東京大学
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Priority claimed from JP2017154566A external-priority patent/JP2020153661A/ja
Application filed by 国立研究開発法人医薬基盤・健康・栄養研究所, 学校法人北里研究所, 国立大学法人東京大学 filed Critical 国立研究開発法人医薬基盤・健康・栄養研究所
Publication of WO2019013256A1 publication Critical patent/WO2019013256A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates generally to the field of evaluating the quality of conserved biological specimens.
  • the present invention relates to a method for evaluating the reliability of stored biological specimens, such as blood samples, plasma samples, serum samples etc., that can withstand the biological evaluation, markers used for the evaluation, It relates to a kit for making the evaluation.
  • Biobank Japan (BBJ: 4-6-1 Shirokanedai, Minato-ku, Tokyo) was established in 2003 in Japan. Since 2003, BBJ has collected and analyzed 51 diseases, 260,000 people and 420,000 cases, focusing on multifactorial diseases, and has built the world's largest disease biobank. Registered cases have 5,837 items of cleaned information and include 95% average follow-up rate and survival information spanning approximately 10 years of average follow-up.
  • the sample DNA, serum, and tissue have a track record of being applicable to omics analysis such as whole genome sequencing, metabolome, proteome, etc.
  • the sample distribution track record is about 16,000 DNA samples and about 10,000 serum samples. However, it is hard to say that the world's largest biobank specimen has been effectively utilized so far. One of the reasons is that the quality control of samples, in particular, the quality control of proteins that are more susceptible to collection and storage than DNA, has not been performed.
  • the present inventors have developed leading-edge proteomics technology to establish a method for developing and measuring a quality evaluation marker for stored serum and plasma samples.
  • Factors that affect the quality of stored blood, serum, and plasma samples include the time from blood collection to centrifugation, storage time and storage conditions for temperature, and the number of freeze-thaw cycles, etc.
  • the present inventors have found that some of the peptides produced by enzymatic treatment of proteins are unstable ones that cause quantitative and qualitative changes under the influence of storage conditions etc. We found that there was something, and focused on the quantitative change of these peptides.
  • the present inventors have found that it is possible to evaluate the quality of a sample by taking the ratio of a stable peptide that is stable in the sample and a variable peptide that is unstable in the sample, and completed the present invention.
  • the present invention includes the following aspects. ⁇ Method for evaluating the quality of stored samples obtained from Biobank etc.> [1] A method for evaluating the quality of a biological sample that has been isolated, wherein a peptide derived from a specific protein produced by treating the sample with an enzyme is used as an index for quality evaluation. [2] Among the peptides derived from the same specific protein in the enzyme-treated sample, the peptides are variable peptides that cause quantitative and / or qualitative changes due to factors affecting quality, and stable peptides that do not cause changes [, 1] The method described.
  • variable peptide and stable peptide derived from the same specific protein can also mean a combination of an individual stable peptide and a variable peptide when there are a plurality of variable peptides and stable peptides, respectively, and It can mean the combination as a set of each of a plurality of variable peptides.
  • variable peptide and the stable peptide derived from the same specific protein are quality evaluation markers shown in the following table: Table 3-1, Table 3-2, Table 3-3, Table 4-1, Table 4-2, Table 4-2, Table 4-3, Table 4-4, Table 5, Table 6, Table 8- described in the present specification. 1, Table 8-2, Table 8-3, Table 8-4, Table 8-5, Table 8-6, Table 8-7, Table 9-1, Table 9-2, Table 9-3, Table 9- 4, Table 9-5, Table 9-6, Table 9-7, Table 10, and Table 11.
  • variable peptide and the stable peptide derived from the same specific protein may be any of the quality evaluation markers derived from the same specific protein shown in the above table, and also the quality evaluation markers derived from the same specific protein shown in the table It may be all. [8] The method according to [7], wherein the variable peptide and the stable peptide derived from the same specific protein are selected from at least one combination of quality evaluation markers shown in parallel in the table.
  • variable peptide and stable peptide are shown in Table 3-1, Table 3-2, Table 3-3, Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 5
  • the variable peptide and the stable peptide derived from the same specific protein may be any of the quality evaluation markers derived from the same specific protein shown in the above table, and also the quality evaluation markers derived from the same specific protein shown in the table It may be all.
  • the method according to [9] wherein the variable peptide and the stable peptide derived from the same specific protein are selected from at least one combination of quality evaluation markers shown in parallel to the table.
  • variable peptide and the stable peptide derived from the same specific protein are quality evaluation markers shown in the following table: Table 13-1, Table 13-2, Table 13-3, Table 13-4, Table 13-5, Table 13-6, Table 13-7, Table 13-8, and Table 13-9 described in the present specification. , Table 13-10, Table 15-1, Table 15-2, Table 15-3, Table 15-4, Table 15-5, Table 15-6, Table 15-7, Table 15-8, Table 17-1. , And Table 17-2.
  • the variable peptide and the stable peptide derived from the same specific protein may be any of the quality evaluation markers derived from the same specific protein shown in the above table, and also the quality evaluation markers derived from the same specific protein shown in the table It may be all.
  • the method according to [11] wherein the variable peptide and the stable peptide derived from the same specific protein are selected from at least one combination of quality evaluation markers shown in parallel in the table.
  • a method of selecting a peptide derived from a protein present in a sample, which can evaluate the quality of a biological sample being isolated comprising: 1) Collect biological samples from animals including humans, 2) Load the biological samples collected with factors that affect their quality, 3) The factor-loaded sample is treated with an enzyme to cleave proteins present in the sample into peptides, 4) Among the obtained peptides, those which cause quantitative and / or qualitative changes due to factors affecting the quality and those which do not cause change are respectively identified as a variable peptide and a stable peptide, 5) Identify combinations of one or more variable peptides derived from the same specific protein and one or more stable peptides, 6) Use the identified combination as a marker that can assess the quality of the biological sample, or 6 ') The method, wherein at least one of the identified combinations is a marker capable of evaluating the quality of a biological sample.
  • a method of producing a marker capable of evaluating the quality of a biological sample being isolated comprising: 1) Collect biological samples from animals including humans, 2) Load the biological samples collected with factors that affect their quality, 3) The factor-loaded sample is treated with an enzyme to cleave proteins present in the sample into peptides, 4) Among the obtained peptides, identify peptides that cause quantitative and / or qualitative changes depending on factors affecting quality, and peptides that do not cause changes; 5) The method, wherein the identified peptide is used as a quality evaluation marker of a biological sample. [19] The method according to [18], wherein a biological sample is collected from healthy human in step 1).
  • a quality evaluation marker which is a variable peptide and a stable peptide derived from the same specific protein shown in the table described in [8] or [11].
  • a quality evaluation marker which is a combination of at least one variable peptide and stable peptide derived from the same specific protein shown in parallel to the table described in [8] or [11].
  • FIG. 1 shows an outline of analysis for identifying quality evaluation markers of the present invention.
  • FIG. 2-1 shows the results of verification of serum peptides by SRM / MRM method, which showed fluctuations with storage temperature and period.
  • the solid line shows the transition of the relative quantitative value of the variable peptide, and the broken line shows the transition of the relative quantitative value of the stable peptide.
  • FIG. 2-2 shows the results of verification of serum peptides by SRM / MRM method, which showed fluctuations with storage temperature and period.
  • the solid line shows the transition of the relative quantitative value of the variable peptide, and the broken line shows the transition of the relative quantitative value of the stable peptide.
  • FIG. 2-3 shows the results of verification of serum peptides by SRM / MRM method, which showed variation with storage temperature and period.
  • FIG. 3-1 shows the results of verification of plasma peptide by SRM / MRM method, in which fluctuation was observed depending on storage temperature and period.
  • the solid line shows the transition of the relative quantitative value of the variable peptide, and the broken line shows the transition of the relative quantitative value of the stable peptide.
  • FIG. 3-2 shows the results of verification of plasma peptide by SRM / MRM method, in which fluctuation was observed depending on storage temperature and period.
  • the solid line shows the transition of the relative quantitative value of the variable peptide, and the broken line shows the transition of the relative quantitative value of the stable peptide.
  • FIG. 3-3 shows the results of verification of plasma peptide by SRM / MRM method, in which fluctuation was observed depending on storage temperature and period.
  • the solid line shows the transition of the relative quantitative value of the variable peptide
  • the broken line shows the transition of the relative quantitative value of the stable peptide.
  • FIG. 3-4 shows the results of verification of plasma peptide by SRM / MRM method, in which fluctuation was observed depending on storage temperature and period.
  • the solid line shows the transition of the relative quantitative value of the variable peptide
  • the broken line shows the transition of the relative quantitative value of the stable peptide.
  • FIG. 4 shows the results of verification of serum peptides by SRM / MRM method in which fluctuations in time from blood collection to centrifugation were observed.
  • FIG. 5 shows the results of verification of plasma peptides by SRM / MRM method which fluctuated in time from blood collection to centrifugation.
  • the solid line shows the transition of the relative quantitative value of the variable peptide, and the broken line shows the transition of the relative quantitative value of the stable peptide.
  • FIG. 6 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions in plasma, at room temperature, and within one week.
  • FIG. 7-1 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of 6 ° C. and 6 months in plasma.
  • FIG. 7-2 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of 4 ° C. and 6 months or less in plasma.
  • FIG. 7-3 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of 4 ° C. and 6 months or less in plasma.
  • FIG. 7-4 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of 4 ° C. and 6 months or less in plasma.
  • FIG. 8-1 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of ⁇ 30 ° C. and 6 months or less in plasma.
  • FIG. 8-2 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of ⁇ 30 ° C. and 6 months or less in plasma.
  • FIG. 9-1 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of -80 ° C. and within 6 months in plasma.
  • FIG. 9-2 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under storage conditions of -80 ° C. and within 6 months in plasma.
  • FIG. 10 shows the results of selection of quality evaluation markers by shotgun quantitative analysis in serum, at room temperature, and under storage conditions within one week.
  • FIG. 11-1 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under serum storage conditions at 4 ° C. and within 6 months.
  • FIG. 11-2 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under serum storage conditions at 4 ° C. and within 6 months.
  • FIG. 12-1 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under serum storage conditions of ⁇ 30 ° C. and 6 months or less.
  • FIG. 12-2 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under serum storage conditions of ⁇ 30 ° C. and 6 months or less.
  • FIG. 13-1 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under serum storage conditions of ⁇ 80 ° C. and within 6 months.
  • FIG. 13-2 shows the results of selection of quality evaluation markers by shotgun quantitative analysis under serum storage conditions of ⁇ 80 ° C. and within 6 months.
  • FIG. 14 shows the results of selection of quality evaluation markers in plasma and after freeze-thawing by shotgun quantitative analysis.
  • FIG. 15 shows the results of selection of quality evaluation markers in serum and after freeze thaw by shotgun quantitative analysis.
  • FIG. 16-1 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in serum under the following storage conditions.
  • AS etc. show a sample number.
  • AS 0 h 0 hours
  • AS 6 m_4 4 ° C., 6 months
  • AS 1 y 4 4 ° C., 1 year
  • AS 6 m 30 -30 ° C., 6 months
  • AS 1 y 30 -30 ° C., 1 year
  • AS 1 y 80: -80 ° C, 1 year AS 1 y 80: -80 ° C, 1 year.
  • FIG. 16-2 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in serum under the following storage conditions.
  • FIG. 16-3 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in serum under the following storage conditions.
  • FIG. 16-4 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in serum under the following storage conditions.
  • FIG. 17-1 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in plasma under the following storage conditions.
  • AP etc. show a sample number.
  • FIG. 17-2 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in plasma under the following storage conditions.
  • FIG. 17-3 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in plasma under the following storage conditions.
  • FIG. 17-4 shows the results of selection of quality evaluation markers by SRM / MRM quantitative analysis in plasma under the following storage conditions.
  • FIG. 18 shows the results of selection of quality evaluation markers in serum and each time until centrifugation by SRM / MRM quantitative analysis.
  • LS etc. show a sample number.
  • LS_CT10 After 10 minutes
  • LS_CT30 30 minutes
  • LS_CT2h After 2 hours
  • LS_CT6h After 6 hours.
  • the present invention is a method for evaluating the quality of an isolated biological sample, wherein a peptide derived from a specific protein produced by treating the sample with an enzyme is used as an indicator of quality evaluation.
  • the method more specifically, the presence of the variable peptide and the stable peptide, wherein the peptide is a variable peptide and a stable peptide among peptides derived from the same specific protein in the enzyme-treated sample It relates to the method, wherein the ratio is an index of quality assessment.
  • assessing the quality of a biological sample means a method of examining whether the quality of a biological sample has been degraded and evaluating whether it can be used with confidence. Do. Quality assessment is very important in that it prevents misdiagnosis caused by test results using specimens with degraded quality.
  • quality of the biological sample refers to the property of the biological sample that the sample is available or not available for the purpose of use of the biological sample.
  • the purpose of use of biological samples is, for example, early diagnosis of various diseases, understanding of pathological conditions, and determination of treatment guidelines by analysis such as genome analysis, transcriptome analysis, proteome analysis, peptidome analysis, metabolomic analysis, etc. is there.
  • biological sample refers to blood, serum, plasma, urine, tissue, saliva, lymph fluid, tissue fluid (inter-tissue fluid, obtained from humans or animals such as pets and livestock). It means a specimen of biological origin selected from intercellular fluid, wet and dry fluid) and body cavity fluid (joint fluid, cerebrospinal fluid, serosal fluid, aqueous humor).
  • blood, serum, plasma, urine, tissues, cerebrospinal fluid which are derived from human beings, are currently stored as being stored in a storage organization.
  • blood, serum, plasma, urine, tissue or cerebrospinal fluid is used as a biological sample, blood is more preferred, and its liquid component, serum or plasma is more preferred. Conventional known methods can be used as methods for preparing serum and plasma.
  • isolated is synonymous with being conserved and is meant to distinguish it from a sample in a living organism.
  • Biobank Japan Minato Ward, Tokyo
  • National Center Biobank Network Shinjuku Ward, Tokyo
  • preservation organizations such as Toyama 1-21-1), but it is not limited to these organizations.
  • the present invention can be applied to any sample such as a biological sample stored in a hospital, a clinic, a laboratory, or the like.
  • enzyme treatment refers to digesting a protein into peptide fragments using an enzyme, which includes, for example, sequence specific proteases, etc. be able to.
  • sequence-specific protease refers to a protease that cleaves a specific peptide bond, and examples thereof include trypsin, pepsin, chymotrypsin, glutamyl endopeptidase, lysyl endopeptidase and the like.
  • the enzyme treatment of the present invention is preferably performed by trypsin or a combination of trypsin and lysyl endopeptidase.
  • a peptide derived from a specific protein means a peptide produced by digesting a protein with an enzyme.
  • the phrase "peptide as an indicator for quality evaluation” means that the type of peptide body, its abundance, and if there are multiple types of peptides, the abundance ratio as an indicator for quality assessment. And “to make the abundance ratio of the variable peptide and the stable peptide an index of quality evaluation” described later.
  • the type of peptide body, its abundance, and the abundance ratio of multiple peptides can be confirmed, for example, by measuring the amount of change qualitatively and / or quantitatively for the peptide by mass spectrometry. Moreover, these can also be confirmed by ELISA using an antibody.
  • variable and stable peptides derived from the same specific protein are, among the peptides derived from the same specific protein, quantitative and / or quality depending on factors affecting the quality. Refers to peptides that cause a change and peptides that do not change, the said quantitative and / or qualitative changes being confirmed by measuring the peptide qualitatively and quantitatively, for example by mass spectrometry. Also, quantitative and / or qualitative changes may be confirmed by ELISA using antibodies.
  • the variable peptide and the stable peptide derived from the same specific protein are selected from the group of digested peptides obtained by enzymatic treatment of a biological sample.
  • variable peptide and a stable peptide derived from the same specific protein can also mean a combination of an individual stable peptide and a variable peptide, when there are a plurality of variable peptides and stable peptides respectively, It can mean the combination as a set of each of the peptide and the plurality of variable peptides. It will be clear to the person skilled in the art which of the two is meant, depending on the situation considered.
  • the abundance ratio of the variable peptide and the stable peptide means the ratio of the variable peptide to the stable peptide in the variable peptide and the stable peptide derived from the same specific protein, that is, the amount of the variable peptide / stable It means the amount of peptide.
  • the abundance ratio can be confirmed, for example, by measuring the amount of change qualitatively and / or quantitatively for the peptide by mass spectrometry. Moreover, these can also be confirmed by ELISA using an antibody. In practice, since the combination of individual stable peptides and variable peptides is complicated and inefficient when compared individually, the average value of the stable peptide of each protein can be compared with the average value of the variable peptides.
  • the threshold value of the abundance ratio for judging that the quality of the biological sample is not deteriorated, ie, the quality is guaranteed, varies depending on the combination of the variable peptide and the stable peptide, ie, the quality evaluation marker. If the abundance ratio of the fluctuating peptide to the stable peptide (amount of fluctuating peptide: amount of stable peptide) is within the threshold, the quality of the biological sample is evaluated as not deteriorating, and if it is outside the threshold , It can be evaluated that the quality has deteriorated.
  • the threshold of the abundance ratio in which the quality has not deteriorated in the quality evaluation marker is, for example, 0.77: 1 to 1.3: 1 (amount of variable peptide: amount of stable peptide), and if it falls within this threshold, It can be evaluated that the quality of the sample has not deteriorated.
  • a "quality assessment marker” is a peptide that combines one or more variable peptides and one or more stable peptides, and is a marker used to evaluate the quality of a biological sample.
  • a “quality assessment marker” is a peptide that combines one or more variable peptides and one or more stable peptides, and is a marker used to evaluate the quality of a biological sample.
  • combinations of variable and stable peptides shown in parallel to the tables described herein.
  • analysis of the peptide includes selected reaction monitoring (SRM).
  • SRM selected reaction monitoring
  • the SRM / MRM method using a mass spectrometer can simultaneously quantify hundreds of different peptides, for example, it is possible to quantify all of the variable and stable peptides shown in parallel in the table herein at one time. .
  • the ratio is calculated using the combination of the variable peptide and the stable peptide in the same quantified protein, the closer the value is to 1, it can be inferred that the protein is more stable.
  • variable peptide and the stable peptide as an index for quality evaluation.
  • those quality evaluation marker peptides are quantified all at once by the SRM / MRM method, and the abundance ratio of the variable peptide and the stable peptide is calculated.
  • the number of combinations of quality evaluation markers whose ratio is between, for example, 0.77 and 1 the quality of the sample is evaluated to be better (preserved state is better).
  • the quality of the sample is evaluated as good. Conversely, the more the number of combinations whose ratio is less than 0.77, the worse the quality of the sample (for example, the storage quality is poor), for example, in the case of 30 out of 50, preferably 50 to 40, more preferably 50 to 45 Evaluate).
  • the present invention is a method of selecting a peptide derived from a protein present in a sample capable of evaluating the quality of a biological sample being isolated, which is quantitative according to factors affecting the quality. And / or provide methods for identifying unstable peptides that cause qualitative changes and stable peptides that do not cause changes.
  • factors affecting quality include storage conditions such as storage temperature and duration, and time until centrifugation.
  • “To cause a quantitative and / or qualitative change” means to change quantitatively and / or qualitatively, and to decrease or increase the quantitative value.
  • Does not cause quantitative and / or qualitative change means that the quantitative value is constant or nearly constant without changing quantitatively and / or qualitatively.
  • Unstable peptides are referred to as "variable peptides" in the present invention.
  • the stable peptide is a peptide selected from the group of peptides derived from the same protein as the protein from which the specific unstable peptide is derived, and a peptide that does not undergo quantitative and / or qualitative changes under storage conditions and the like. It is.
  • Stable peptides are referred to as “stable peptides" in the present invention.
  • the quantitative value of a certain peptide in serum stored in the biobank is lower than the average value of healthy people, the quantitative value at the start of storage is unknown, so the quantitative value decreases during storage It is not possible to distinguish if the subject who collected the sample had a value lower than the average value of the healthy subject.
  • a stable peptide is used as a control, it can be distinguished whether the decrease in the quantitative value is the effect of storage or individual differences among subjects. Because the quantitative value of the stable peptide should not change before and after storage, it reflects the state before storage.
  • Each combination of variable and stable peptides according to the invention is composed of peptides derived from the same specific protein.
  • the quality of the biological sample can be evaluated without being affected by individual differences in the protein in the biological sample.
  • factor affecting quality refers to a factor that causes a change in the amount and / or quality of the sample, and can include, for example, storage conditions of the sample.
  • the storage conditions of the sample include, for example, storage temperature of the sample, storage period, time from collection of sample for storage of sample such as centrifugation to preparation, number of times of freezing and thawing of sample, and the like.
  • the storage temperature of the specimen for example, room temperature (20 to 30 ° C.), refrigeration (0 to 10 ° C.), freezing (-20 to 40 ° C.), ultra low temperature (-60 to -90 ° C.), liquid nitrogen etc. may be mentioned.
  • the storage period is, for example, 0 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 40 minutes, 60 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours. Hours, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 6 months, 6 months, 12 months, 18 months, 24 months, 30 months , 36 months, etc.
  • the time from sample collection to preparation for sample storage is 0 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 30 minutes, 40 minutes, 60 minutes, 90 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 6 hours , 8 hours, 12 hours, 24 hours, etc. can be mentioned.
  • As the number of times of freezing and thawing of the sample for example, 0 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times and the like can be mentioned.
  • the method for detecting and quantifying the quantitative and / or qualitative change of the peptide may be any method capable of specifically detecting the variable peptide and the stable peptide, and may include, for example, mass spectrometry.
  • mass spectrometry a peptide sample is converted to a gaseous ion (ionization) using an ion source, and in the analysis unit, the peptide sample is ionized by moving in vacuum and using electromagnetic force, or ionized according to the time of flight difference.
  • a separation method such as an ion cyclotron resonance type can be appropriately selected.
  • tandem mass spectrometry combining two or more mass spectrometry methods and triple quadrupole mass spectrometry can be used.
  • the sample when the sample is a sample containing a phosphorylated peptide, the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) prior to sample introduction into the mass spectrometer.
  • Fe-IMAC iron ion-immobilized affinity chromatography
  • the variable peptide and the stable peptide according to the present invention can be separated and purified by liquid chromatography (LC) or HPLC to obtain a sample.
  • the detection unit and the data processing method can be selected appropriately.
  • the stable isotope labeled peptide is a stable isotope labeled peptide in which one or more of the amino acids in the variable peptide and the stable peptide according to the present invention contains any one or more of 15N, 13C, 18O, and 2H.
  • the type, position, number, etc. of amino acids can be appropriately selected, and the stable isotope-labeled peptide can be selected from the F-moc method (Amblard., Et al. Methods Mol Biol. Using an amino acid labeled with a stable isotope).
  • mass spectrometry that detects and quantitates quantitative and / or qualitative changes in peptides is SRM / MRM using stable isotope-labeled synthetic peptides.
  • Selected reaction monitoring is a quantitative analysis method using a triple quadrupole mass spectrometer and uses an analysis system coupled to a liquid chromatograph. When multiple molecules are targeted for measurement, it is called multiple reaction monitoring (MRM).
  • Mass spectrometry combining selective reaction monitoring (SRM) and multiple reaction monitoring (MRM) has high quantitativity and selectivity, and can quantify the target protein in the sample.
  • mass spectrometry for detecting and quantifying quantitative and / or qualitative changes in a peptide is an analytical method using an isotope tag or an isobaric tag.
  • isotope tags include dimethyl labeling reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark) reagent and the like
  • isobaric tags include TMT reagent (registered trademark) And iTRAQ (registered trademark) reagent etc. can be mentioned.
  • Peptides derived from proteins in serum and plasma can be comprehensively analyzed by analytical methods using isotope tags or isobaric tags.
  • the analysis method is a candidate for a combination of a variable peptide and a stable peptide (quality evaluation marker) which can be used for quality evaluation of a biological specimen that satisfies a certain standard.
  • the combination of the selected variable peptide and the stable peptide can be used to further narrow down the quality evaluation markers by verifying whether the combination of the selected variable peptides and stable peptides can be further used for the quality evaluation of biological samples by the above-mentioned SRM / MRM method.
  • the method for detecting and quantifying the quantitative and / or qualitative change of a peptide may include a pretreatment step of obtaining the peptide from a biological sample prior to mass spectrometry.
  • a pretreatment step of obtaining the peptide from a biological sample prior to mass spectrometry for example, protein extraction from a biological sample, digestion of a protein into a peptide with an enzyme or the like, desalting / concentration of a peptide, etc. can be mentioned.
  • the present invention provides, in one aspect, a method of screening for peptides derived from proteins present in a sample, which can assess the quality of the biological sample being isolated.
  • the method is 1) collecting a biological sample from an animal including human; 2) loading a factor that affects the quality of the collected biological sample, 3) treating the sample loaded with the factor with an enzyme to divide proteins present in the sample into peptides, 4) identifying, among the obtained peptides, peptides that cause quantitative and / or qualitative changes due to factors affecting quality and those that do not cause changes as variable peptides and stable peptides, respectively; 5) identifying a combination of one or more variable peptides derived from the same specific protein and one or more stable peptides; 6) using the identified combination as a quality evaluation marker of a biological sample, or 6 ′)
  • a step of making at least one of the specified combinations a quality evaluation marker of a biological sample may be included.
  • 1) collecting a biological sample from an animal including human comprises collecting a biological sample selected from blood, serum, plasma, urine and tissue.
  • Animals, including humans include humans as well as companion animals such as dogs and cats and livestock such as cows and pigs.
  • it is a human being, preferably a healthy human being.
  • the biological sample is serum or plasma.
  • the step of loading the collected biological sample with a factor affecting its quality is the above-mentioned time from blood collection to centrifugation, storage as a factor affecting the quality It may include temperature, storage period and number of freeze-thaw cycles.
  • treating the factor-loaded sample with an enzyme and dividing the protein present in the sample into peptides comprises enzymatic treatment with trypsin or a combination of trypsin and lysyl endopeptidase
  • the enzyme treatment is with trypsin.
  • a peptide that causes a quantitative and / or qualitative change depending on factors affecting quality and a peptide that does not cause a change respectively, a variable peptide and a stable peptide
  • identifying the combination of one or more variable peptides derived from the same specific protein and one or more stable peptides is an isotopic tag or isobaric described herein. It may include mass spectrometry using tags and SRM / MRM identification using synthetic peptides with stable isotope labels.
  • the step of identifying the variable peptide and the stable peptide performs comprehensive analysis (shotgun quantitative proteomics) of the peptide using TMT labeling or dimethyl labeling, and the variable peptide and the stable peptide are determined based on certain criteria. It may involve selecting peptide candidates, followed by selection of variable peptides and stable peptide candidates suitable for target proteomics (SRM / MRM analysis), and further performing SRM / MRM analysis using stable isotope-labeled peptides.
  • comprehensive analysis shotgun quantitative proteomics
  • Selection criteria for peptides of variation peptide candidates from shotgun quantitative proteomics results include meeting one or more of the following: (1) The quantitative value of each condition fluctuates by 1.3 or more times the quantitative value of the control sample (sample at the start of storage); (2) Among the peptides having the variation of (1), the variation is common to a certain number of subjects among a plurality of subjects, for example, 7 to 8 out of 10 subjects, 5 to 6 out of 8 subjects , Common to 3 out of 4 subjects; (3) The quantitative value fluctuates as time passes.
  • the variation of the quantitative value may be 2.0 times or more, 1.7 times or more, 1.5 times or more, or 1.3 times or more.
  • the criteria may include meeting one or more of the following: (1) a peptide having a sequence unique to a protein; (2) Does not contain a cleavage error by trypsin; (3) Among the same proteins, shotgun quantitative proteomics has identified peptides with less variation (stable peptides).
  • the criteria it is also optional to add the criteria "it does not contain the amino acid (methionine) containing the unsuitable modification in quantification ;;" between the above-mentioned criteria (1) and (2).
  • the specified combination, or 6 ') at least one of the specified combinations is a quality evaluation marker of a biological sample
  • the step of validating a marker candidate by SRM / MRM analysis May be included.
  • the step of setting it as the quality evaluation marker is a step of SRM / MRM analyzing the variable peptide and the stable peptide candidate selected in step 5) above and setting a peptide meeting certain criteria as the quality evaluation marker. obtain.
  • Selection criteria of the variable peptide in the quality evaluation marker by SRM / MRM analysis include satisfying any one or more of the following: (1) The quantitative value of each condition fluctuates by 1.3 or more times the quantitative value of the control sample (sample at the start of storage); (2) Among the peptides having the variation of (1), the variation is common to a certain number of subjects among a plurality of subjects, for example, 7 to 8 out of 10 subjects, 5 to 6 out of 8 subjects , Common to 3 out of 4 subjects. In a preferred embodiment, the variation of the quantitative value may be 2.0 times or more, 1.7 times or more, 1.5 times or more, or 1.3 times or more.
  • the quality evaluation markers of the biological specimen obtained as described above are, for example, the quality evaluation markers shown in the table herein as the variable peptide and the stable peptide derived from the same specific protein. And at least one combination of
  • the present invention provides, in one aspect, a method of producing a quality assessment marker for a biological sample that has been isolated.
  • the method is 1) collecting a biological sample from an animal including human; 2) loading a factor that affects the quality of the collected biological sample, 3) treating the sample loaded with the factor with an enzyme to divide proteins present in the sample into peptides, 4) identifying, among the obtained peptides, peptides that cause quantitative and / or qualitative changes depending on factors affecting quality, and peptides that do not cause changes; 5) It may include the step of using the identified peptide as a quality evaluation marker of a biological sample.
  • 1) collecting a biological sample from an animal including human comprises collecting a biological sample selected from blood, serum, plasma, urine and tissue.
  • Animals, including humans include humans as well as companion animals such as dogs and cats and livestock such as cows and pigs.
  • it is a human being, preferably a healthy human being.
  • the biological samples are serum and plasma.
  • the step of loading the collected biological sample with a factor affecting its quality is the above-mentioned time from blood collection to centrifugation, storage as a factor affecting the quality It may include temperature, storage period and number of freeze-thaw cycles.
  • treating the factor-loaded sample with an enzyme and dividing the protein present in the sample into peptides comprises enzymatic treatment with trypsin or a combination of trypsin and lysyl endopeptidase
  • the enzyme treatment is with trypsin.
  • the step of identifying can comprise mass spectrometry using isotopic or isobaric tags as described herein and identification by SRM / MRM using synthetic peptides with stable isotope labels.
  • the step of identifying as a variable peptide and a stable peptide performs comprehensive analysis (shotgun quantitative proteomics) of the peptide using TMT labeling or dimethyl labeling, and based on certain criteria, the variable peptide and the stable peptide are stable. It may involve selecting peptide candidates, followed by selection of variable peptides and stable peptide candidates suitable for target proteomics (SRM / MRM analysis), and further performing SRM / MRM analysis using stable isotope-labeled peptides.
  • Selection criteria for peptides of variation peptide candidates from shotgun quantitative proteomics results include meeting one or more of the following: (1) The quantitative value of each condition fluctuates by 1.3 or more times the quantitative value of the control sample (sample at the start of storage); (2) Among the peptides having the variation of (1), the variation is common to a certain number of subjects among a plurality of subjects, for example, 7 to 8 out of 10 subjects, 5 to 6 out of 8 subjects , Common to 3 out of 4 subjects; (3) The quantitative value fluctuates as time passes.
  • the variation of the quantitative value may be 2.0 times or more, 1.7 times or more, 1.5 times or more, or 1.3 times or more.
  • the criteria may include meeting one or more of the following: (1) a peptide having a sequence unique to a protein; (2) Does not contain a cleavage error by trypsin; (3) Among the same proteins, shotgun quantitative proteomics has identified peptides with less variation (stable peptides).
  • the criteria it is also optional to add the criteria "it does not contain the amino acid (methionine) containing the unsuitable modification in quantification ;;" between the above-mentioned criteria (1) and (2).
  • the step of using the identified peptide as a quality evaluation marker of a biological sample may include verification of a marker candidate by SRM / MRM analysis.
  • the step of setting it as the quality evaluation marker is a step of SRM / MRM analyzing the variable peptide and the stable peptide candidate selected in step 5) above and setting a peptide meeting certain criteria as the quality evaluation marker. obtain.
  • Selection criteria of the variable peptide in the quality evaluation marker by SRM / MRM analysis include satisfying any one or more of the following: (1) The quantitative value of each condition fluctuates by 1.3 or more times the quantitative value of the control sample (sample at the start of storage); (2) Among the peptides having the variation of (1), the variation is common to a certain number of subjects among a plurality of subjects, for example, 7 to 8 out of 10 subjects, 5 to 6 out of 8 subjects , Common to 3 out of 4 subjects. In a preferred embodiment, the variation of the quantitative value may be 2.0 times or more, 1.7 times or more, 1.5 times or more, or 1.3 times or more. In SRM / MRM analysis performed on a partially narrowed peptide group that is shotgun quantitative proteomics, peptides can be widely selected by setting the quantitative value fluctuation to 1.3 times or more.
  • the quality assessment markers of biological specimens produced as described above are shown, for example, as variable peptides and stable peptides derived from the same specific protein in parallel in the table herein. And at least one combination of quality assessment markers.
  • the quality evaluation marker of the present invention includes other quality evaluation markers manufactured by the variation of the conditions described in the above-mentioned respective steps, and is not limited to the quality evaluation markers described in the table in the present specification. It is.
  • the present invention is a variable peptide and stable peptide derived from the same specific protein shown in the table herein as a quality evaluation marker capable of evaluating the quality of the biological specimen being isolated.
  • a quality assessment marker that can be used to assess the quality of the biological specimen being isolated, in combination and at least one combination of variable peptide and stable peptide from the same specific protein shown in parallel in the table herein
  • the present invention also provides, in one embodiment, an isolated biological comprising a variable peptide and a stable peptide derived from the same specific protein as shown in the Table herein, including stable isotopes.
  • kits are a combination of variable and stable peptides, ie, stable isotope labeled peptides corresponding to quality assessment markers, as well as separate protein solubilization reagents and protein digestion reagents, enzyme treatment reagents, etc. Or in a single container.
  • the kit may also be equipped with instructions for assessing the quality of the biological sample.
  • the kit of the present invention comprises a stable isotope labeled peptide of a quality assessment marker, which stable isotopically labeled peptide detects quantitative and / or qualitative changes in the peptide as described herein. It may be a peptide suitable for mass spectrometry to be quantified.
  • the present inventors In order to select and produce quality evaluation markers, the present inventors first changed the time to centrifugation of serum and plasma of 4 healthy persons, storage temperature and duration, and the number of freeze-thaw cycles to Identification of the unstable peptide to be received was performed using shotgun quantitative proteomics. Time to centrifugation is 15 minutes to 6 hours, Storage temperature and duration are room temperature, 4 ° C, -30 ° C, -80 ° C, several hours to 6 months with liquid nitrogen, freeze / thaw 1 to 5 times The effects of centrifugation conditions, storage conditions and freeze-thaw conditions were examined up to the number of times.
  • Serum and plasma were collected from 4 healthy persons and collected under various storage conditions (storage temperature, storage period, time from blood collection to centrifugation, number of freeze-thaw cycles).
  • the serum and plasma samples were searched for candidate peptides for quality evaluation markers by two types of shotgun quantitative proteomics, and among the peptides that varied the marker candidate peptides for storage temperature and period and for each time from blood collection to centrifugation, Selected.
  • verification experiments by target proteomics SRM / MRM method using stable isotope-labeled peptides
  • SRM / MRM method using stable isotope-labeled peptides are performed for selected marker candidate peptides, and the identified marker candidate peptides are certainly varied among the storage conditions of the sample. It was confirmed that it was or was stable.
  • the outline of the analysis which identified the quality evaluation marker is shown in FIG.
  • Example 1 Shotgun quantitative proteomics of serum and plasma samples 1
  • Example 1.1 Recovery Conditions of Serum and Plasma Specimens and Protein Extraction and Digestion from the Specimens Blood for four healthy subjects is collected, separated as serum and plasma respectively, and specimens are collected for each condition of storage temperature and storage period did. In addition, samples separated by time until centrifugation after blood collection are also collected. The sample recovery conditions are shown in Table 2.
  • Protein extraction and digestion from serum and plasma samples Protein extraction from serum and plasma samples stored under each condition and digestion to peptide fragments with digestive enzymes were performed by phase transfer extraction (PTS method: Phase transfer surfactant) (Ref. 4). The procedure is shown below. After dissolving each sample with MPEX PTS reagent (GL Science, Tokyo, Japan), incubate at 95 ° C for 10 minutes, add dithiothreitol to a final concentration of 5 mM or TCEP to a final concentration of 33.3 mM, and perform a reduction reaction for 30 minutes Further, iodoacetamide was added to a final concentration of 20 mM or 53 mM to carry out the alkylation reaction.
  • PTS method Phase transfer surfactant
  • Example 1.2 Shotgun comparative quantitative analysis using TMT labeling of digested peptides
  • the digested peptides prepared in Example 1.1 were stable isotope labeled with Tandem Mass Tag (TMT) reagent (Thermo Scientific, Bremen, Germany) After that, 7 fractions were performed with a C18-SCX StageTip column (Reference 6). Thereafter, each fraction was subjected to shotgun quantitative proteome analysis by LC-MS / MS.
  • TMT Tandem Mass Tag
  • Mass spectrometer Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany), liquid chromatography: UltiMate 3000 Nano-flow high-performance LC (HPLC) system (Dionex, Sunnyvale, CA), autosampler: HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland). Further, for the introduction of the sample into the mass spectrometer, a self-made analytical column in which a C18-AQ resin of 1.9 ⁇ m was sealed in a needle with an inner diameter of 75 ⁇ m and a length of 300 mm was used.
  • the mobile phase of LC was constituted by mobile phase A (0.1% formic acid and 2% acetonitrile) and mobile phase B (0.1% formic acid and 90% acetonitrile).
  • the sample is dissolved in buffer A and loaded on a trap column (0.075 x 20 mm, Acclaim PepMap RSLC Nano-Trap Column; Thermo Scientific), then the mobile phase is 5-35 in 120 minutes at a flow rate of 280 nL / min of LC. Unfolded with a slope of% B.
  • the conditions for measurement of Full MS by Q-Exactive were as follows. Scan range: 350-1800 m / z, resolution: 70000, ion integration: 3 ⁇ 10 6 .
  • the MS / MS measurement conditions were as follows.
  • Example 1.3 Shotgun Comparative Quantitative Analysis of Digested Peptide Using Dimethyl Labeling The digested peptide of each sample prepared in Example 1.1 is divided into two, one of which is a stable isotope labeled dimethylation reagent (DM-H). The other was labeled with unlabeled dimethylating reagent (DM-L) (Ref. 7).
  • the reagents for dimethyl labeling used were formaldehyde P / N 252549 (Sigma Aldrich, St.
  • the configuration of the LC-MS / MS apparatus was as follows. Mass spectrometer: LTQ-Orbitrap Discoverer (Thermo Scientific), liquid chromatography: Nanospace SI-2 system (Shiseido, Tokyo). Further, for the introduction of the sample into the mass spectrometer, a capsule pack C18 MGIII-H column (Shiseido) with an inner diameter of 2.0 mm and a length of 50 mm was used.
  • the mobile phase of LC was constituted by mobile phase A (0.01% formic acid) and mobile phase B (0.01% formic acid and 90% acetonitrile). The samples were developed at a mobile phase flow rate of 200 ⁇ L / min, with a gradient of 0-27% (70 minutes), 27-55% (14 minutes).
  • the conditions for Full MS measurement by LTQ-Orbitrap Discoverer were as follows. Scan range: 400-2000 m / z, resolution: 30000, ion integration 5 x 105.
  • the MS / MS measurement conditions were as follows. Top 5 precursor ion, maximum ion time: 200 ms, ion integration: 105, ion selection threshold: 103, separation width: 2 Da.
  • Identification analysis of the measured RAW data file was performed by SEQUEST Search (Thermo Scientific) on Proteome Discoverer V3.1 (Thermo Scientific), and the database used UniProt human protein database. The threshold value of the identified protein was set to a precursor mass error range of 3 ppm and a fragment ion mass error range of 0.8 Da.
  • Identification proteins and peptides were determined with a Proteome Discoverer standard setting with a percent positive rate (FDR) of less than 1%.
  • the comparison analysis for the Full MS spectrum used LC-MS analysis software Skyline Ver. 3.6 (MacCoss Lab.). Comparative analysis by XIC (extracted-ion chromatogram) was performed using a peptide library identified by repeating similar measurement in advance.
  • peptides serving as marker candidates were selected. Among the total quantification peptides, those whose relative quantitative value with the control (0 hour) sample fluctuates 1.5 times or more for each condition are regarded as the peptide that has changed, and the change is common to 3 out of 4 people The candidate peptides were identified as Furthermore, peptides with inconsistent changes in the amount of peptide over time were excluded from candidates. These candidate peptides were selected for each serum and plasma, and were also selected for each condition of storage temperature and period and time after blood collection and centrifugation. The candidate marker that satisfies these conditions was 738 peptide (196 protein).
  • Example 2 LC-SRM / MRM Analysis
  • SRM / MRM analysis was performed according to the method previously reported (Ref. 8).
  • a peptide stabilized by PTS method is dissolved in a 2% acetonitrile solution containing 0.1% trifluoroacetic acid (TFA), and a synthetic stable isotope labeled peptide having the same sequence as the target peptide as an internal standard (SpikeTide L; JPT Peptide Technologies , Berlin, Germany). It was then analyzed using a triple quadrupole mass spectrometer coupled with nanoflow LC.
  • the configuration of the device was as follows.
  • Triple quadrupole mass spectrometer TSQ-Vantage (Thermo Fisher Scientific, Bremen, Germany), Nanoflow LC: Paradigm MS2 (Michrom BioResources, Auburn, CA), Autosampler: HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland) .
  • the sample was introduced into a mass spectrometer using a self-made analytical column in which a 1.9 ⁇ m C18-AQ resin was sealed on a needle with an inner diameter of 75 ⁇ m and a length of 100 mm.
  • the mobile phase of LC consisted of buffer A (0.1% formic acid and 2% acetonitrile) and B (0.1% formic acid and 90% acetonitrile).
  • the mobile phase is 5 to 45 min at a flow rate of 280 nL / min of LC. It was developed with a 35% B gradient.
  • the analysis in the SRM mode was performed under the following conditions. Q1 Peak Width: 0.7 FWHM, Cycle time: 1 sec, Collision Gass Pressure: 1.8 mTorr. The collision energy was optimized for each SRM transition, and the intensity of each transition was measured in schedule mode with a peak duration of 5 minutes.
  • SRM / MRM Target Peptide and Transition Evaluation As a target peptide of a biomarker candidate protein, peptides having a sequence suitable for SRM / MRM analysis were selected from peptides identified by shotgun proteomics. The criteria for selection were as follows. (1) A sequence unique to a protein (2) An amino acid (methionine) containing an inappropriate modification for quantification (methionine) is not included (3) A miss by cleavage with trypsin is not included.
  • transitions of SRM / MRM measurement of each peptide (combination of m / z of parent ion and daughter ion) were prepared from MS / MS spectrum library of shotgun proteomics using Skyline software (MacCoss Lab) .
  • the measurement transition of the selected peptide is performed by first selecting eight strong transitions per peptide, and performing SRM analysis of the selected peptide on the peptide prepared from the pooled serum, and finally the signal to noise ratio (S / N)
  • the peptide with three or more transitions whose>is> 10 was selected.
  • confirmation of whether or not the selected transition can correctly detect the target peptide can be made by using a stable isotope-labeled peptide (SItable) having the same sequence as the signal of the endogenous target peptide and the target peptide added as an internal standard. It was judged by the similarity of the peak area ratio in the transition with the peptide). This similarity is represented by "dotp" in Skyline software.
  • the SI-peptide was synthesized as isotopically labeled C-terminal Arg13C6; 15N4 or Lys 13C6; 15N2 heavy peptide (SpikeTide L, JPT Peptide Technologies, Berlin, Germany) (crude purity).
  • the quantitative value of target peptide by SRM / MRM was calculated as the peak area ratio (Peak Area Ratio) of the SI-peptide added to each sample as an internal standard and the endogenous target peptide .
  • the RAW data file output as a result of the measurement by the mass spectrometer was analyzed using Skyline software (freeware; MacCoss Lab.) To calculate the peak area detection and the area ratio.
  • variable peptide candidates and stable peptide candidates were identified.
  • those with higher peak intensity for identification were selected.
  • the number of candidate stable and variable peptides selected from these criteria was a total of 356 peptides.
  • results in shotgun quantitative proteome analysis were verified by SRM / MRM method.
  • a stable isotope-labeled peptide having the same sequence as the selected peptide was synthesized, and a synthetic peptide mix was added as an internal standard to serum / plasma samples for four as in shotgun quantitative proteome analysis.
  • SRM / MRM analysis was performed on the samples for each storage condition, and the quantitative value of each sample was compared with the quantitative value of the control sample as in the shotgun quantitative proteome analysis.
  • the criteria for verification of the fluctuation peptide candidate in the SRM / MRM method were 1.3 or more times the fluctuation of the quantitative value, and were common to 3 out of 4 persons. At the same time, it was confirmed that the stable peptide also did not fluctuate in the quantitative value, and in the same protein, a combination of the variable peptide and the stable peptide could be verified as a quality evaluation marker.
  • the marker candidates satisfying these criteria are 14 sets of serum variable peptides as stored at each storage temperature (Table 3-1, Table 3-2 and Table 3-3, and FIGS. 2-1, 2-2). And Figure 2-3), 32 sets of plasma (Table 4-1, Table 4-2, Table 4-3 and Table 4-4 and Figure 3-1, Figure 3-2, Figure 3-3 and Figure 3- 3 4) There was.
  • Example 3 Shotgun quantitative proteomics and SRM / MRM quantitative analysis of serum and plasma samples 2
  • Candidate peptides for quality evaluation markers were selected according substantially to the procedure described in Example 1 except that the recovery conditions in the following table.
  • Example 4 Quality evaluation of biological samples using quality evaluation markers
  • quality evaluation markers In order to evaluate the quality of biological samples such as plasma or serum stored in storage institutions such as Biobank Japan, Table 3-1 to Table 6 and one or more combinations of quality assessment markers listed in Tables 8-1 to 11.
  • the quality evaluation marker peptides described in Tables 3-1 to 6 and Tables 8-1 to 11 are quantified for the obtained plasma or serum samples using SRM / MRM analysis.
  • the combination of the quality evaluation marker of the peptide and the stable peptide is 21 respectively There are 29 types.
  • SRM / MRM analysis is performed on the peptides of these quality evaluation markers all at once, and the ratio of variable peptide to stable peptide is calculated.
  • SRM / MRM analysis is performed on the peptides of these quality evaluation markers all at once, and the ratio of variable peptide to stable peptide is calculated.
  • Example 5 SRM / MRM quantitative analysis of serum samples
  • Candidate peptides for quality evaluation markers were selected substantially according to the procedure described in Example 1 except that recovery conditions in the following table.
  • any of the individual stable peptides can be combined with the individual variable peptides. Also, any of the individual variable peptides can be combined with the individual stable peptide. See Figures 16-1 and 16-2.
  • any of the individual stable peptides can be combined with the individual variable peptides. Also, any of the individual variable peptides can be combined with the individual stable peptide. See Figures 16-1 and 16-2.
  • any of the individual stable peptides can be combined with the individual variable peptides. Also, any of the individual variable peptides can be combined with the individual stable peptide. See Figures 16-3 and 16-4.
  • any of the individual stable peptides can be combined with the individual variable peptides. Also, any of the individual variable peptides can be combined with the individual stable peptide. See Figures 16-3 and 16-4.
  • any of the individual stable peptides can be combined with the individual variable peptides. Also, any of the individual variable peptides can be combined with the individual stable peptide. See Figures 16-3 and 16-4.
  • Example 6 SRM / MRM quantitative analysis of plasma samples
  • Candidate peptides for quality evaluation markers were selected substantially in accordance with the procedure described in Example 1 except that the recovery conditions in the following table.
  • any of the individual stable peptides can be combined with the individual variable peptides. Also, any of the individual variable peptides can be combined with the individual stable peptide. See Figures 17-1 and 17-2.
  • any of the individual stable peptides can be combined with the individual variable peptides. Also, any of the individual variable peptides can be combined with the individual stable peptide. See Figures 17-1 and 17-2.
  • Example 7 SRM / MRM quantitative analysis of serum samples by time to centrifugation The serum is used according to the procedure substantially as described in Example 1, except that the recovery conditions of the table below are the recovery conditions.
  • Candidate peptides for quality evaluation markers were selected according to time, 10 minutes, 30 minutes, 2 hours and 6 hours.
  • any one of the individual stable peptides can be combined with each individual variable peptide.
  • any of the individual variable peptides can be individually set with each individual stable peptide. It can be put together.
  • any one of the individual stable peptides can be combined with the individual variable peptides. Either can be combined.) ”. This meaning is described, for example, with reference to Table 13-5 below.
  • Table 13-5 means the following nine combinations: Above, the same expressions are interpreted similarly.

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Abstract

L'invention concerne un procédé d'analyse grâce auquel la qualité d'un échantillon de sérum ou de plasma stocké peut être évaluée de manière très précise et rapide, ledit procédé comprenant l'identification d'un marqueur de protéine ou de peptide pouvant être utilisé pour l'évaluation et l'utilisation d'une protéomique quantitative. Dans ledit procédé, utilisé pour évaluer la qualité d'un échantillon biologique isolé, un peptide dérivé d'une protéine spécifique, qui est synthétisé par traitement de l'échantillon à l'aide d'une enzyme, est utilisé en tant qu'indicateur de l'évaluation de qualité.
PCT/JP2018/026202 2017-07-11 2018-07-11 Procédé d'évaluation de la qualité d'un échantillon biologique et marqueur associé WO2019013256A1 (fr)

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