WO2019013256A1

WO2019013256A1 - Quality evaluation method for biological specimen and marker therefor

Info

Publication number: WO2019013256A1
Application number: PCT/JP2018/026202
Authority: WO
Inventors: 毅朝長; 白水　崇; 義男小寺; 七里　眞義; 晃一郎湯地
Original assignee: 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
Priority date: 2017-07-11
Filing date: 2018-07-11
Publication date: 2019-01-17

Abstract

Provided is an assay method whereby the quality of a stored serum or plasma specimen can be highly accurately and quickly evaluated, said method comprising identifying a protein or peptide marker that can be used for the evaluation and using quantitative proteomics. In this method, which is used for evaluating the quality of an isolated biological specimen, a peptide derived from a specific protein, which is synthesized by treating the specimen with an enzyme, is used as an indicator of the quality evaluation.

Description

Method for evaluating quality of biological sample and marker therefor

The present invention relates generally to the field of evaluating the quality of conserved biological specimens. In particular, 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.

In order to realize personalized medicine, it is necessary to systematically collect specimens for a large number of diseases and comprehensively analyze the relationship between diseases and genes and proteins. To that end, 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.

For quality control of samples stored in storage institutions such as Biobank Japan, in order to evaluate the quality, it is necessary to compare two samples immediately after collection or before storage and after storage. However, in Biobank Japan etc., only samples after storage are stored, and samples immediately after collection can not exist. Therefore, it is necessary to carry out the quality evaluation only on the specimen after storage.

There is no international standard for the indicator of quality evaluation of proteins in biological samples that have been stored in Biobank Japan etc. so far, and there is no such thing as an international standard, and only a few documents are seen (patent documents 1, non-patent documents 1 to 3). However, these measurement methods can not be said to be sufficient for the quality evaluation of proteins in biological samples.

Special table 2017-512996

Therefore, there is a need for a reliable new indicator for conducting the quality evaluation of samples stored in Biobank Japan and the like. Therefore, 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. We identified protein and peptide markers that can be evaluated, and used quantitative proteomics to establish a measurement method that can be evaluated with high accuracy and speed.

As described above, in Biobank Japan etc., since there is no sample immediately after collection, it is necessary to carry out the quality evaluation only with the sample after storage. And, when evaluating the quality of the sample only after storage, it is a problem to interpret whether the difference in a certain index in the sample is caused by the storage condition, the time until centrifugation, etc., or it is caused by the individual difference, etc. ing.
That is, originally, the amount of protein in the blood is different among individuals, so if quantitative change of protein in the sample is used as an indicator for quality evaluation of stored samples, is it due to quality deterioration or individual difference? That is, it can not be distinguished between inter-individual variation and intra-individual variation. Therefore, 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. Here, for the first time, 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.

Therefore, 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. The “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.

[3]
The method according to [1] or [2], wherein the abundance ratio of the variable peptide and the stable peptide derived from the same specific protein is used as an index for quality evaluation.
[4]
The method according to any one of [1] to [3], wherein the abundance ratio of the variable peptide and the stable peptide derived from the same specific protein is calculated using the respective averages of the variable peptide and the stable peptide derived from the same specific protein . When there are a plurality of “the same variable protein and a plurality of variable peptides derived from a specific protein”, it means that the average of the plurality of stable peptides and the plurality of variable peptides as a set is calculated. In addition, when one of “the same variation protein and stable peptide derived from the specific protein” is one and the other is present in a plurality, it also includes the case of calculating the average value of one value of one and the plurality of other values. .
[5]
The method according to any one of [1] to [4], wherein the enzyme treatment is performed by trypsin.
[6]
The method according to any one of [1] to [5], wherein the biological sample is selected from blood, serum, plasma, urine, tissue and cerebrospinal fluid.

[7]
The method according to any one of [1] to [6], wherein the 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. 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.
[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.

[9]
The same specific protein-derived 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 method according to claim 7, which is a quality evaluation marker shown in Table 6 and Table 6. 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.
[10]
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.
[11]
The method according to [7], wherein the 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.
[12]
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.

<Method of sorting variable peptides and stable peptides>
[13]
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.
[14]
The method according to [13], wherein a biological sample is collected from healthy human in step 1).
[15]
The method according to [13] or [14], wherein the enzyme treatment is performed by trypsin.
[16]
The method according to any of [13] to [15], wherein the biological sample is selected from blood, serum, plasma, urine, tissue, cerebrospinal fluid.
[17]
The method according to any one of [13] to [16], wherein the factor affecting quality is selected from time from blood collection to centrifugation, storage temperature, and storage period.

<Method for producing variable peptide and stable peptide>
[18]
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).
[20]
The method according to [18] or [19], wherein the enzyme treatment is performed by trypsin.
[21]
The method according to any one of [18] to [20], wherein the biological sample is selected from blood, serum, plasma, urine, tissue and cerebrospinal fluid.
[22]
The method according to any one of [18] to [21], wherein the factor affecting quality is selected from time from blood collection to centrifugation, storage temperature, and storage period.

<Invention related to the combination of peptides used to evaluate the quality of samples obtained from Biobank etc.>
[23]
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].
[24]
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].
[25]
Use of a variable peptide and a stable peptide derived from the same specific protein shown in the table described in [8] or [11] as a quality evaluation marker capable of evaluating the quality of an isolated biological sample.
[26]
Quality evaluation that can evaluate the quality of the biological specimen being isolated in the combination of at least one of the variable peptide and the stable peptide derived from the same specific protein shown in parallel to the table described in [8] or [11] Use as a marker.
[27]
Assess the quality of the biological analyte being isolated, which contains the variable peptide and the stable peptide derived from the same specific protein shown in the table described in [8] or [11] and containing stable isotopes. Kit to do.
[28]
An isolated organism comprising at least one of a variable peptide and a stable peptide derived from the same specific protein shown in parallel in the table described in [8] or [11], which contains a stable isotope. For evaluating the quality of biological samples.

At present, the importance of personalized medicine and precision medicine (Precision Medicine) is being called out at home and abroad, but in order to realize this, analysis with multiple specimens collected at multiple facilities is essential. The need is increasing day by day. In fact, biobanks have been established at university hospitals in various places in Japan, and many samples are beginning to be collected. In order to effectively use the blood and urine samples stored there, it is essential to evaluate the quality of the samples. The invention of the method for evaluating the quality of a sample, the quality evaluation marker, the method for producing a quality evaluation marker, etc., which we found this time, exerts great power in the quality control of a sample regardless of whether it is domestic or foreign. According to the present invention, it is possible to obtain an objective evaluation result without depending on individual differences among specimens.

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. 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. 3-1 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, and 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, and 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. 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. 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. In addition, 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 6 m 80: -80 ° C., 6 months, 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. In addition, AP etc. show a sample number. AP0h: 0 hour, AP6m_4: 4 ° C, 6 months, AP1y_4: 4 ° C, 1 year, AP6m_30: -30 ° C, 6 months, AP1y_30: -30 ° C, 1 year, AP6m_80: -80 ° C, 6 months, AP1y_80: -80 ° C, 1 year. 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. In addition, 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.

As used herein, "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. Here, "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.

As used herein, the term "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). At present, in practice, blood, serum, plasma, urine, tissues, cerebrospinal fluid, which are derived from human beings, are currently stored as being stored in a storage organization. Preferably, 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.

As used herein, "isolated" is synonymous with being conserved and is meant to distinguish it from a sample in a living organism. As an organization where samples are stored, Biobank Japan (Minato Ward, Tokyo), Tohoku University Tohoku Medical Megabank Organization (1-1, Seiryocho, Aoba Ward, Sendai City), National Center Biobank Network (Shinjuku Ward, Tokyo) There are preservation organizations such as Toyama 1-21-1), but it is not limited to these organizations. For example, 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.

As used herein, "enzyme treatment" or "treating with an enzyme" refers to digesting a protein into peptide fragments using an enzyme, which includes, for example, sequence specific proteases, etc. be able to. The 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.

As used herein, "a peptide derived from a specific protein" means a peptide produced by digesting a protein with an enzyme.

As used herein, 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.

The “variable and stable peptides derived from the same specific protein” as used herein 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. In addition, “a 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.

As used herein, "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.

As used herein, 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. . For example, combinations of variable and stable peptides shown in parallel to the tables described herein. However, in practice, since the combination of individual stable peptides and variable peptides becomes complicated and inefficient when compared individually, it is possible to compare the average value of the stable peptide of each protein with the average value of the variable peptides.

In the method of the present invention, wherein the quality of a biological sample being isolated is evaluated, and a peptide derived from a specific protein is used as an indicator for quality evaluation, analysis of the peptide includes selected reaction monitoring (SRM). Mass spectrometric SRM / MRM method combining multiple reaction monitoring (MRM) and is preferable. 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. . When 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. The more such proteins, the better the quality of the sample used for quantification.
In the following, the evaluation using the abundance ratio of the variable peptide and the stable peptide as an index for quality evaluation will be exemplified.
For example, if there are 50 combinations of variable peptide and stable peptide quality evaluation markers, 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. As 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). For example, if the ratio is between 0.77 and 1 in a combination of 30, 50, preferably 50 to 40, more preferably 50 to 45 out of 50, 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).

In another aspect, 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. Examples of the "factor 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. As a factor which makes a peptide unstable, for example, a change in mass number due to decomposition or modification by protease can be mentioned. In addition, 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.

As in the present invention, in selecting "a variable peptide" and "a stable peptide" to evaluate the quality of a biological sample, let us evaluate the quality of the biological sample by quantitative values of only unstable peptides. If the quantitative value of the peptide fluctuates depending on the storage conditions such as storage temperature, duration, time to centrifugation, or individual differences among subjects from which the sample was collected, that is, due to inter-individual fluctuation or intra-individual fluctuation This is because the quality of the target biological sample can not be evaluated because it can not be determined. More specifically, when 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. Thus, if 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. That is, when stable peptides are at the same level as the average value of healthy people, but unstable peptides decrease, it can be judged that the decrease in quantitative value of unstable peptides is the effect of storage. Therefore, by using the stable peptide as a control, when the stable peptide is the same quantitative value as the average value of healthy people even after storage, the decrease of the quantitative value of the unstable peptide can be determined due to storage. On the other hand, when both quantitative values of stable peptide and unstable peptide after storage are constant values lower than the average value of healthy persons, it can be judged that the decrease in quantitative value of unstable peptide is due to individual difference.

Each combination of variable and stable peptides according to the invention is composed of peptides derived from the same specific protein. When the variable peptide and the stable peptide in a specific combination are derived from the same protein, the quality of the biological sample can be evaluated without being affected by individual differences in the protein in the biological sample. In practice, it is complicated and inefficient to compare combinations of individual stable peptides and variable peptides individually, so it is possible to compare the average value of the stable peptide of each protein with the average value of the variable peptides.

As used herein, "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. As 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. Preferably 25 ° C. ± 2 ° C., 4 ° C. ± 3 ° C., −30 ° C. ± 5 ° C., −80 ° C. ± 8 ° C. and in liquid nitrogen. 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.

In the present invention, 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. . In 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. This is a measurement method using a mass spectrometer that can separate and detect according to the ratio, and as a method of ionization using an ion source, EI method, CI method, FD method, FAB method, MALDI method, ESI method Methods such as magnetic field deflection type, quadrupole type, ion trap type, time of flight (TOF) type, Fourier transform as methods of separating ionized peptide samples in the analysis section. A separation method such as an ion cyclotron resonance type can be appropriately selected. In addition, tandem mass spectrometry (MS / MS) combining two or more mass spectrometry methods and triple quadrupole mass spectrometry can be used. In addition, 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. In addition, 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. Also, the detection unit and the data processing method can be selected appropriately. When detecting and quantifying a variable peptide and a stable peptide by mass spectrometry using mass spectrometry, using a stable isotope-labeled peptide having a known concentration and consisting of the same amino acid sequence as the peptide as an internal standard it can. 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). 298: 3-24 (2005)), which can be chemically synthesized, but iTRAQ (R) reagent, ICAT (R) reagent, ICPL (R) reagent, NBS (R) reagent It can also be prepared using a labeling reagent such as Tandem Mass Tag (TMT) (registered trademark) reagent.

In one embodiment of the present invention, 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 (SRM) 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.

In another embodiment of the present invention, 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. Examples of isotope tags include dimethyl labeling reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark) reagent and the like, and examples of 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. In another embodiment of the present invention, 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.

In one embodiment of the present invention, 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. As the pretreatment, 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.

<Method of sorting variable peptides and stable peptides>
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.

In one embodiment of the present invention, 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. Specifically, it is a human being, preferably a healthy human being. Also preferably, the biological sample is serum or plasma.

In one embodiment of the present invention, 2) 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.

In one embodiment of the present invention, 3) 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 Preferably, the enzyme treatment is with trypsin.

In one embodiment of the present invention, 4) among the obtained peptides, 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 And 5) 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. In a preferred embodiment, 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.

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.
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. Since shotgun quantitative proteomics selects peptides to a certain extent exhaustively, in order to narrow down to an appropriate number of peptides, quantitative value fluctuation of not less than 1.3 times but not less than 1.5 times, 1.7 times or more, 2.0 times or more is adopted appropriately Do.

As selection criteria for peptides suitable for SRM / MRM analysis, 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). In addition, 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).

In one embodiment of the present invention, 6) 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. In a preferred embodiment, 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 one embodiment of the present invention, 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

<Method for producing variable peptide and stable peptide>
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.

In one embodiment of the present invention, 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. Specifically, it is a human being, preferably a healthy human being. Also preferably, the biological samples are serum and plasma.

In one embodiment of the present invention, 4) among the obtained peptides, peptides that cause quantitative and / or qualitative changes depending on factors affecting quality (variable peptides) and peptides that do not cause changes (stable peptides) 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. In a preferred embodiment, 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.

In one embodiment of the present invention, 5) 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. In a preferred embodiment, 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.

<Invention related to the combination of peptides used for quality evaluation of samples obtained from Biobank etc.>
In one embodiment of the present invention, 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. It should be noted that 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.

In addition, in one embodiment, 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. As 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 On the use of

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. A kit for assessing the quality of an analyte, and at least one of a variable peptide and a stable peptide derived from the same specific protein shown in parallel in the table herein, containing stable isotopes, A kit is provided to assess the quality of the biological sample being isolated. As used herein, a “kit” is 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. Preferably, 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.

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.
As a result, comprehensive proteome analysis of serum and plasma identified about 6,000 kinds of peptides, and among them, hundreds of unstable peptides affected by centrifugation conditions, storage conditions and freeze-thaw conditions were identified. There were stable peptide groups in the proteins from which these peptides were derived.
Next, the candidate peptides of these quality evaluation markers were verified by target proteomics using SRM / MRM method. As a result, a total of 98 types were identified as fluctuating peptides that produced quantitative and / or qualitative changes under the influence of the time until centrifugation and storage conditions, and stable peptides that did not produce quantitative and / or qualitative changes. .
EXAMPLES The present invention will be described in detail by way of examples, but it should be noted that these do not limit the scope of the present invention and are merely illustrative.

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. In addition, verification experiments by target proteomics (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. Then, 1% (w / w) trypsin (proteomics grade; Roche Mannheim, Germany) or 2% (w / w) lysyl endopeptidase (Wako Pure Chemical Industries, Osaka, Japan) and 2% (w / w) to the sample Trypsin (Promega Corporation, Madison, USA) was added to digest the extracted protein for 12 hours at 37 ° C. After digestion, equal volumes of ethyl acetate and 1% final concentration of trifluoroacetic acid were added and vortexed to separate the surfactant in the solution into the organic phase. After centrifugation, the aqueous phase containing the peptide was recovered and desalted by Stage Tips to obtain a digested peptide (Reference 5).

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. The configuration of the LC-MS / MS apparatus was as follows. 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 μm was sealed in a needle with an inner diameter of 75 μm 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. Top 10 precursor ions, injection time: 120 ms, resolution: 35000, MS / MS ion selection threshold: 5 × 10 ⁴ counts, separation width: 3.0 Da. Analysis of the measured RAW data file was performed by MaxQuant software (Ver 1.5.1.2). Search engine Andromeda, UniProt human protein database was used. The threshold value of the identified protein was set to a precursor mass error range of 7 ppm and a fragment ion mass error range of 0.01 Da. Identification proteins and peptides were determined with a false positive rate (FDR) of less than 1% against the reverse database.

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. Louis, USA), stable isotope labeled formaldehyde P / N 596388 (Sigma Aldrich), sodium cyanoborohydride P / N 714435 (Sigma) Aldrich), cyano deuterated sodium borate P / N SC 258 163 (Sigma Aldrich). Thereafter, DM-H in which all samples to be compared were mixed was mixed in DM-L of each sample as an internal standard. Thereafter, acetonitrile was added to a final concentration of 5%, and then TFA was added to a final concentration of 1% to precipitate a surfactant in the solution, and the supernatant was collected as a sample to obtain a sample for LC-MS / MS. 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 μL / 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.

(Result of Example 1)
Search for quality evaluation markers by shotgun quantitative proteomics Serum and plasma samples collected from 4 healthy subjects are placed under conditions of storage temperature, storage period, and time from blood collection to centrifugation, then shot by TMT method and dimethyl labeling method Conditional variation peptides were searched by cancer quantitative proteome analysis. The quantitative value of each sample was calculated as a relative peak area (Peak Area Ratio) where the peak area of four control samples (sample digested immediately after blood collection) was 1. Peptides were collected from a total of 272 samples under all storage conditions of serum and plasma, and stable isotope labeling was performed (TMT labeling: 336 runs of 48 sets × 7 fractions, 327 runs of 56 sets of dimethyl labels). As a result of analysis of 663 runs in total, 5905 peptides (865 proteins) were identified in total.

Selection of Marker Candidate Peptides from Shotgun Quantitative Analysis Results From all identified peptides for which quantitative values were calculated, 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 The method of 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 μm C18-AQ resin was sealed on a needle with an inner diameter of 75 μm 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). After dissolving the sample in buffer A and loading it on a trap column (0.075 x 20 mm, Acclaim PepMap RSLC Nano-Trap Column; Thermo Scientific), 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.

Selection of 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. In addition, 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. Furthermore, 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. And dotp> 0.9 was set to the threshold value of endogenous peptide detection. 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).

Quantification of target peptide using internal standard peptide 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.

(Result of Example 2)
Selection of Marker Candidate Peptides for SRM / MRM Analysis Next, among the marker candidate peptides selected according to the sample conditions, peptides suitable for SRM / MRM analysis were narrowed down. The selection criteria were those in which the peptide sequence was protein specific, did not contain methionine, and did not contain any trypsin cleavage errors. In addition, among the same proteins, those having identified peptides (stable peptides) which showed little fluctuation in shotgun quantitative proteome analysis were considered as candidates. Also for the stable peptides, those meeting the same selection criteria as the variable peptide candidates were selected. Among the same protein, for those in which a plurality of 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.

Verification of Marker Candidates in SRM / MRM Analysis For selected 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. After that, 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. Furthermore, there were 12 pairs of serum (Table 5 and FIG. 6) and 9 pairs of plasma (Table 6 and FIG. 5) as peptides which fluctuate with time from blood collection to centrifugation. The peptides tested under all these storage conditions totaled 100 peptides (34 proteins). In addition, the peptide which overlapped between storage conditions was counted as 1.

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.

The obtained results are as shown in the following table. The figure number which shows the result of having selected the quality evaluation marker by shotgun quantitative analysis and SRM / MRM quantitative analysis is also shown simultaneously under various conditions.

P00450
Ceruloplasmin
143
NNEGTYYSPNYNPQSR
141
ALYLQYTDETFR
P00450
Ceruloplasmin
143
NNEGTYYSPNYNPQSR
297
EVGPT NADPVCLAK
P00450
Ceruloplasmin
143
NNEGTYYSPNYNPQSR
55
GAYPLSIEPIGVR
P00450
Ceruloplasmin
143
NNEGTYYSPNYNPQSR
140
DLYSGLIGPLIVCR

Example 4: Quality evaluation of biological samples using 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.
Variation of Serum_Temp in Table 3-1 to Table 3-3 or Plasma_Temp (quality evaluation marker of storage condition of serum or plasma) in Table 4-1 to Table 4-4 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. The higher the number of combinations of quality evaluation markers whose ratio is between 0.77 and 1, the better the quality of the sample is. Conversely, the more the number of combinations whose ratio is less than 0.77, the worse the quality of the sample is.
Furthermore, there are 12 kinds and 8 kinds of combinations of quality evaluation markers of fluctuation peptide and stable peptide, respectively, of Serum_CT of Table 5 or Plasma_CT (marker of time from blood sampling of blood or plasma to centrifugation) of Table 6. 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. The greater the number of combinations of quality assessment markers whose ratio is between 0.77 and 1.3, the shorter the time to centrifugation of the sample. Conversely, the more the number of combinations whose ratio is less than 0.77 or 1.3 or more, the longer the time to centrifugation of the sample.

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.

The obtained results are as shown in the following table. Put together for each specific protein from which the variable and stable peptides are derived.

See Figures 16-1 and 16-2.

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.

See Figures 16-1 and 16-2.

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 16-3 and 16-4.

See Figures 16-3 and 16-4.

See Figures 16-3 and 16-4.

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.

The obtained results are as shown in the following table. The specific peptides from which the variable and stable peptides are derived are summarized.
See Figures 17-1 and 17-2.

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.

See Figures 17-1 and 17-2.

See Figures 17-3 and 17-4.

See Figures 17-3 and 17-4.

See Figures 17-3 and 17-4.

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.

The obtained results are as shown in the following table. The specific peptides from which the variable and stable peptides are derived are summarized.
See FIG.

See FIG.

In the above example, “In the following table, any one of the individual stable peptides can be combined with each individual variable peptide. Also, any of the individual variable peptides can be individually set with each individual stable peptide. It can be put together. " Also, in the claims, “but in the above table, 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.

References
1. Rai AJ et al., HUPO Plasma Proteome Project sample collection and handling: Towards the standardization of parameters for plasma proteome samples. Proteomics, 5: 3262-3277 (2005).
2. Elliott P et al., The UK Biobank Sample handling and storage protocol for the collection, processing and archiving of human blood and urine. International Journal of Epidemiology, 37: 234-244 (2008).
3. Hubel A et al., Storage of Human Biospecimens: Selection of the Optimal Storage Temperature. BIOPRESERVATION AND BIOBANKING, 12: 1-11 (2014).
4. Masuda, T., Tomita, M. & Ishihama, Y. Phase transfer surfactant-aided trypsin digestion for membrane proteinome analysis. J. Proteome Res. 7, 731-740 (2008).
5. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896-1906 (2007).
6. Adachi, J. et al. Improved proteinome and phosphoproteome analysis on a cation exchanger by a combined acid and salt gradient. Anal. Chem. 88, 7899-7903 (2016).
Stable-isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75, 6843-6852, 2003. 7. J. L. Hsu, S. Y. Huang, N. H. Chow, S. H. Chen.
8. Kume, H. et al. Discovery of color cancer cancer candidates by membrane proteomics analysis and subsequent verification using selected reaction monitoring (SRM) and tissue microarray (TMA) analysis. Mol. Cell. Proteomics 13, 147-1484 (2014) .

In order to properly realize personalized medicine and precision medicine (precision medicine), analysis of multiple specimens collected at many facilities such as Biobank Japan is essential. Quality assessment of specimens is essential for effective use of specimens stored there. The invention of the method for evaluating the quality of a sample, the quality evaluation marker, the method for producing a quality evaluation marker, etc., which we found this time, exerts great power in the quality control of a sample regardless of whether it is domestic or foreign. In the future, by consigning the measurement from domestic and foreign clinical laboratory companies, it is thought that the international competitiveness of the Japanese pharmaceutical industry will be strengthened, leading to the revitalization of the medical economy.

Claims

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.
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. Item 2. The method according to Item 1.
The method according to claim 1 or 2, wherein the abundance ratio of the variable peptide and the stable peptide derived from the same specific protein is used as an index for quality evaluation.
The method according to any one of claims 1 to 3, wherein the abundance ratio of the variable peptide and the stable peptide derived from the same specific protein is calculated using an average of each of the variable peptide derived from the same specific protein and the stable peptide.
5. The method according to any one of claims 1 to 4, wherein the enzyme treatment is performed by trypsin.
The method according to any one of claims 1 to 5, wherein the biological sample is selected from blood, serum, plasma, urine, tissue and cerebrospinal fluid.
The method according to any one of claims 1 to 6, wherein the variable peptide and the stable peptide derived from the same specific protein are the quality evaluation markers shown in the following table:
The method according to claim 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.
The same specific protein-derived 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 method according to claim 7, which is a quality evaluation marker shown in Table 6.
The method according to claim 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 in the table.
The method according to claim 7, wherein the variable peptide and the stable peptide derived from the same specific protein are selected from among at least one combination of quality evaluation markers shown in the following table:

(However, in the above table, any one of individual stable peptides can be combined with each individual variation peptide. Also, any individual variation peptide can be combined with each individual stable peptide. .)

(However, in the above table, any one of individual stable peptides can be combined with each individual variation peptide. Also, any individual variation peptide can be combined with each individual stable peptide. .)

(However, in the above table, any one of individual stable peptides can be combined with each individual variation peptide. Also, any individual variation peptide can be combined with each individual stable peptide. .)

(However, in the above table, any one of individual stable peptides can be combined with each individual variation peptide. Also, any individual variation peptide can be combined with each individual stable peptide. .)

(However, in the above table, any one of individual stable peptides can be combined with each individual variation peptide. Also, any individual variation peptide can be combined with each individual stable peptide. .)

(However, in the above table, any one of individual stable peptides can be combined with each individual variation peptide. Also, any individual variation peptide can be combined with each individual stable peptide. .)

(However, in the above table, any one of individual stable peptides can be combined with each individual variation peptide. Also, any individual variation peptide can be combined with each individual stable peptide. .)
The method according to claim 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 to 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) The method, wherein the identified combination is a quality evaluation marker of a biological sample.
The method according to claim 13, wherein in step 1), a biological sample is collected from healthy human subjects.
The method according to claim 13 or 14, wherein the enzyme treatment is performed by trypsin.
The method according to any one of claims 13 to 15, wherein the biological sample is selected from blood, serum, plasma, urine, tissue and cerebrospinal fluid.
The method according to any one of claims 13 to 16, wherein the factor affecting quality is selected from time from blood collection to centrifugation, storage temperature, and storage period.
A method of producing a quality assessment marker for an isolated biological sample, 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.
The method according to claim 18, wherein in step 1), a biological sample is collected from healthy human subjects.
20. The method according to claim 18 or 19, wherein the enzyme treatment is performed by trypsin.
21. The method according to any of claims 18 to 20, wherein the biological sample is selected from blood, serum, plasma, urine, tissue, cerebrospinal fluid.
The method according to any one of claims 18 to 21, wherein the factor affecting quality is selected from time from blood collection to centrifugation, storage temperature, and storage period.
A quality evaluation marker which is a variable peptide and a stable peptide derived from the same specific protein shown in the table of claim 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 in the table according to claim 8 or 11.
Use as a quality evaluation marker which can evaluate the quality of the biological specimen isolated in the variation peptide and stable peptide derived from the same specific protein shown by the table of Claim 8 or 11.
A quality evaluation marker capable of evaluating the quality of an isolated biological specimen in at least one combination of a variable peptide and a stable peptide derived from the same specific protein shown in parallel to the table according to claim 8 or 11. Use of.
12. To assess the quality of the biological analyte being isolated, comprising variable peptides and stable peptides derived from the same specific protein as indicated in the table according to claim 8 or 11, comprising stable isotopes. Kit.
12. An isolated biological comprising at least one of a variable peptide and a stable peptide derived from the same specific protein, shown in parallel in the table according to claim 8 or 11, comprising stable isotopes. A kit for evaluating the quality of specimens.