CN116609451A - Quality control product for quality control in thyroid nodule mass spectrometry detection process - Google Patents
Quality control product for quality control in thyroid nodule mass spectrometry detection process Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G01—MEASURING; TESTING
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
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- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/046—Thyroid disorders
Abstract
The application mainly relates to a quality control product for quality control in a thyroid nodule mass spectrometry detection process, which comprises the following components: extracting a protein solution prepared by a cell line with thyroid cancer; and a quality control peptide; the quality control peptide is a peptide segment with specific number of amino acids added at the N end and the C end of the peptide segment sequence, and the first position which is started at the N end of the peptide segment sequence and can be replaced by a heavy-label isotope adopts heavy-label amino acids. The quality control product can be used for judging whether the whole thyroid nodule detection process has errors or not, and can quantify the errors, and the quality control product has good uniformity and stability and small quality control deviation at the reference interval of clinical samples, so that the process of evaluating the malignancy degree or probability of the thyroid nodule by a mass spectrometry is more accurate.
Description
Technical Field
The application mainly relates to the technical field of mass spectrometry detection, in particular to a quality control product for quality control in a thyroid nodule mass spectrometry detection process.
Background
Thyroid Nodules (thyromid Nodules) are a common clinical condition with a prevalence of about 50-60% in the general population, and are common to women and elderly people. Most thyroid nodule patients have no clinical symptoms, often found by physical examination or self-touching, and only 5-15% of thyroid nodules found by pathological examination are malignant nodules, i.e. thyroid cancer.
The currently common thyroid nodule examination method comprises the following steps: serological examination, nuclide scanning, ultrasonic diagnosis, fine needle puncture cytology examination, thyroid needle aspiration cytology examination (FNAC), cervical X-ray examination, thyroid function measurement, etc., however, examination of thyroid nodules by liquid biopsy (such as blood) or genetic testing cannot achieve both good specificity and sensitivity.
The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is widely used for quantitative detection of specific protein markers due to the advantages of high sensitivity, good specificity and the like, but unfortunately, no commercial and mature detection kit capable of evaluating the malignancy or probability of thyroid nodules exists on the market at present, and the detection requirements of high accuracy and high precision cannot be met in the process of evaluating the malignancy or probability of thyroid nodules.
The prior art has the research results of the inventor, such as the Chinese patent of application publication number CN115436640A, discloses a thyroid cancer source cell line CAL62 cell line, 8305C cell line and protein matrix thereof as a substitute matrix of polypeptide capable of evaluating the malignancy or probability of thyroid nodule, has controllable matrix uniformity, can be prepared conventionally, has controllable quality and good intercommunication, can be used for preparing reference substances in a system, a model and a kit for evaluating the malignancy or probability of thyroid nodule, and has high application and auxiliary diagnosis potential.
However, in the quantitative determination process of the protein in the thyroid gland FNA sample by using the LC-MS/MS technology, the benign and malignant degree of the thyroid nodule can be obtained after a series of operations such as cracking, reduction, alkylation, enzymolysis, desalination, on-machine, model determination and the like are required for the protein in the biological sample. This process is complex to operate, has a large number of reagent steps, and is not reproducible in clinical biological samples (FNA). The final result can be affected under the conditions of personnel operation errors, improper reagent storage failure and unstable equipment. However, the same set of detection methods may have different results in different measurement procedures (different people, different equipment, reagents of different storage conditions), where errors may be caused by various aspects such as equipment instability, personnel operating errors, reagent failure due to improper storage, etc.
The foregoing background knowledge is intended to assist those of ordinary skill in the art in understanding the prior art that is closer to the present application and to facilitate an understanding of the inventive concepts and aspects, and it should be understood that the foregoing background art should not be used to assess the novelty of the inventive concepts that lie in the absence of explicit evidence that such disclosure is already disclosed at the time of filing of this patent application.
Disclosure of Invention
In order to solve at least one technical problem mentioned in the background art, the application aims to provide a quality control product for quality control in a thyroid nodule detection process by using a mass spectrometry, which can be used for judging whether an error exists in the whole thyroid nodule detection process, and has the advantages of good uniformity and stability, small quality control deviation and more accurate evaluation of the malignancy degree or probability of the thyroid nodule by using the mass spectrometry, along with a reference section of a clinical sample.
A quality control article for quality control of a thyroid nodule mass spectrometry detection process, comprising:
extracting a protein solution prepared by a cell line with thyroid cancer; and
a quality control peptide;
the quality control peptide is prepared by adding a specific number of amino acids to the N end and the C end of a polypeptide peptide fragment sequence, and adopting a heavy-standard amino acid at the position which is started at the N end of the polypeptide peptide fragment sequence and can be replaced by a heavy-standard isotope.
In some preferred embodiments, the peptide fragment length of the polypeptide peptide fragment is 7 to 38 amino acids, the isoelectric point pI is 3.77 to 9.99, and the hydrophobicity analysis GRAVY value is-1.66 to 1.41.
In some preferred embodiments, the quality control further comprises a diluent.
In some preferred embodiments, the diluent comprises any one of 0.05 to 0.5v% formic acid aqueous solution, 30 to 60v% acetonitrile aqueous solution, 30 to 60v% methanol aqueous solution, or LB solution.
In some preferred embodiments, the theoretical content of the quality control peptide in the quality control product is between the reference interval of the corresponding peptide fragment in the clinical sample.
In some preferred embodiments, the thyroid cancer extraction cell line comprises at least one of a CAL62 cell line, 8305C cell line, BCPAP cell line, 8505C cell line, BHT101 cell line, HTCC3 cell line, or K5 cell line.
In a partially preferred embodiment, the protein solution prepared with the thyroid cancer extraction cell line is in particular prepared at 8X 10 4 The cells were mixed with 5. Mu.L of LB solution by sonication.
In some preferred embodiments, the quality control product comprises at least one high concentration quality control product and at least one low concentration quality control product according to the content of the quality control peptide.
In some preferred embodiments, the preparation process of the quality control product comprises:
1) Adding a diluent into the quality control peptide powder to dilute to obtain an intermediate solution;
2) Mixing the intermediate solution with the protein solution and the diluent according to the proportion to obtain the quality control product.
In a part of the preferred embodiments, the step 1) includes:
1.1 Adding a diluent into the quality control peptide powder for dilution to obtain peptide section mother liquor;
1.2 Adding a diluent into the peptide fragment mother liquor to dilute to obtain an intermediate liquor.
The same set of detection methods may result in different measurement results in different measurement procedures, and errors may be caused by various factors including, but not limited to, equipment instability, personnel operation errors, reagent failure due to improper storage, and the like; in addition, in the process of quantitatively measuring protein/polypeptide in a thyroid gland FNA sample by utilizing liquid chromatography-tandem mass spectrometry LC-MS/MS so as to reflect the benign and malignant degree of a thyroid nodule, the protein in a biological sample can obtain the result of the benign and malignant degree of the thyroid nodule after a series of operations such as cracking, reduction, alkylation, enzymolysis, desalination, on-machine, model measurement and the like. Based on the above, the quality control product for quality control in the thyroid nodule mass spectrometry detection process is provided, and currently, mass spectrometry IVD products for detecting the benign and malignant degree of thyroid nodules are not available.
The quality control product is applied to quality control in thyroid nodule mass spectrometry detection process.
In some preferred embodiments, the thyroid nodule mass spectrometry detection aims to accurately acquire polypeptide mass spectrometry data and/or polypeptide content derived from thyroid nodules.
In some preferred embodiments, the peptide fragment length of the polypeptide is 7 to 38 amino acids, the isoelectric point pI is 3.77 to 9.99, and the GRAVY value of the hydrophobicity assay is-1.66 to 1.41.
In some preferred embodiments, the use comprises:
s1, respectively taking a high-concentration quality control product and a low-concentration quality control product, respectively sequentially carrying out pretreatment, adding an internal standard peptide and three-person three-batch LC-MS/MS detection to obtain a high-concentration quality control product peptide fragment assignment detection content and a low-concentration quality control product peptide fragment assignment detection content, and taking a mean value +/-2 SD of each peptide fragment assignment detection content as an assignment range;
s2, performing pretreatment, adding internal standard peptide, and detecting by an LC-MS/MS method on the FNA sample with red blood cells removed to obtain mass spectrum data of each peptide segment in the FNA sample;
s3, synchronously carrying out step S2, namely respectively taking a high-concentration quality control product and a low-concentration quality control product, and respectively carrying out pretreatment, adding an internal standard peptide and detecting by an LC-MS/MS method to obtain the detection content of each peptide segment in the quality control product;
s4, comparing the detection content of each peptide segment obtained in the step S3 with the assignment range of each peptide segment obtained in the step S1, and if at least one peptide segment does not accord with the assignment result, making errors in the processing process of the FNA samples in the batch;
s5, if the detection content of each peptide segment obtained in the step S3 is within the assignment range of each peptide segment obtained in the step S1, the FNA sample pretreatment and detection process in the step S2 are correct, and a sample result is output;
in some preferred embodiments, the pretreatment, addition of internal standard peptide, and detection by LC-MS/MS method in steps S1, S2, and S3 are all the same.
In some preferred embodiments, the step of pretreatment comprises cleavage, reduction, alkylation, enzymatic hydrolysis, and desalting.
Errors in FNA sample processing include, but are not limited to, personnel errors, equipment errors, reagent errors, etc., which can result in unreliable end results. In the process of processing the FNA sample, the benign and malignant degree of the thyroid nodule can be obtained after a series of operations such as cracking, reduction, alkylation, enzymolysis, desalination, on-machine, model measurement and the like are required to be carried out on proteins in the biological sample, the process is complex in operation, the steps of the reagent are numerous, the clinical biological sample is not renewable, and the final result is influenced by factors such as incorrect storage, invalid operation of personnel, unstable equipment and the like; according to the scheme, the pretreatment and detection are simultaneously carried out by applying the high-low concentration quality control product, and the processing accuracy of the thyroid FNA sample can be indirectly fed back by monitoring the change condition of the quality control product along with the processing process, so that whether the whole thyroid nodule detection process has errors can be judged, the misjudgment rate is reduced, errors caused by misjudgment are avoided, and the final detection accuracy is improved.
In some preferred embodiments, the internal standard peptide is synthesized by using a heavy standard amino acid at the last amino acid at the C-terminal based on the sequence of the peptide fragment to be detected.
In some preferred embodiments, the content of the quality control peptide in the high-concentration quality control product and the low-concentration quality control product is between the reference interval of the corresponding peptide fragment in the FNA sample.
A high quality control thyroid nodule mass spectrometry method comprising:
s1, respectively taking a high-concentration quality control product and a low-concentration quality control product, respectively sequentially carrying out pretreatment, adding an internal standard peptide and three-person three-batch LC-MS/MS detection to obtain a high-concentration quality control product peptide fragment assignment detection content and a low-concentration quality control product peptide fragment assignment detection content, and taking a mean value +/-2 SD of each peptide fragment assignment detection content as an assignment range;
s2, performing pretreatment, adding internal standard peptide, and detecting by an LC-MS/MS method on the FNA sample with red blood cells removed to obtain mass spectrum data of each peptide segment in the FNA sample;
s3, synchronously carrying out step S2, namely respectively taking a high-concentration quality control product and a low-concentration quality control product, and respectively carrying out pretreatment, adding an internal standard peptide and detecting by an LC-MS/MS method to obtain the detection content of each peptide segment in the quality control product;
s4, if the detection content of each peptide segment obtained in the step S3 is within the assignment range of each peptide segment obtained in the step S1, the FNA sample pretreatment and detection process in the step S2 are correct, and a sample result is output;
the pretreatment, the addition of internal standard peptide and the detection operation steps of the upper LC-MS/MS method in the steps S1, S2 and S3 are the same.
The pretreatment of the step comprises the steps of cracking, reduction, alkylation, enzymolysis and desalting.
The LC-MS/MS method is widely used for quantitative detection of specific protein markers due to the advantages of high sensitivity, good specificity and the like, however, no commercial and mature detection kit capable of evaluating the malignancy or probability of thyroid nodules exists on the market at present, and the detection requirement of the LC-MS/MS method with high accuracy and high precision cannot be met in the process of evaluating the malignancy or probability of thyroid nodules. Particularly, the protein in the biological sample detected by the LC-MS/MS method can obtain the result of the benign and malignant degree of the thyroid nodule after a series of operations such as cracking, reduction, alkylation, enzymolysis, desalination, on-machine, model measurement and the like. According to the method, the FNA sample and the high-concentration quality control product are subjected to the same pretreatment and on-machine detection respectively to obtain the quality control product content, and the quality control product content is compared and analyzed with the quality control product content range obtained by independently carrying out assignment detection on the quality control product, so that the quality control can be carried out in the detection process, whether the thyroid nodule detection process has errors or not can be judged on the whole, and the errors which possibly exist can be quantified, so that the detection process is strictly standardized, and the process and the result for evaluating the malignancy degree or probability of the thyroid nodule by a mass spectrometry are more accurate.
The beneficial effects of the application are as follows:
1) The quality control product can be used for judging whether the whole thyroid nodule detection process has errors or not and quantifying the errors possibly existing in the whole thyroid nodule detection process, and can give consideration to the reference interval of clinical samples, so that the quality control product not only reduces the matrix effect, but also solves the adsorption problem of quality control peptide, and ensures the stability and accuracy of the quality control product; the difference between the quality control product and the sample is reduced to a certain extent. The quality control product provided by the application has the advantages of simple preparation method, good uniformity and stability, small quality control deviation and good application prospect.
2) The quality control product is used for processing simultaneously, and the processing accuracy of the thyroid gland FNA sample can be indirectly fed back by monitoring the change condition of the quality control product along with the processing process, so that whether the whole thyroid nodule detection process has errors or not can be judged, the misjudgment rate is reduced, errors caused by misjudgment are avoided, and the final detection accuracy is improved.
3) The quality control product content is obtained after the FNA sample is subjected to pretreatment and on-machine detection with the high-concentration quality control product and the low-concentration quality control product respectively, the quality control product content is compared with the quality control product content range obtained by carrying out assignment detection on the quality control product alone, the quality control can be carried out in the detection process, whether the thyroid nodule detection process has errors or not can be judged on the whole, and errors which possibly exist can be quantified, so that the detection process is strictly standardized, and the process and the result for evaluating the malignancy degree or probability of the thyroid nodule by a mass spectrometry are more accurate.
Drawings
To make the above and/or other objects, features, advantages and examples of the present application more comprehensible, the accompanying drawings which are needed in the detailed description of the present application are simply illustrative of the present application and other drawings can be obtained without inventive effort for those skilled in the art.
FIG. 1 is a flow chart of a quality control product preparation process;
fig. 2 is a flow chart of a mass spectrometry detection process.
Detailed Description
Suitable substitutions and/or modifications of the process parameters will be apparent to those skilled in the art from the disclosure herein, however, it is to be expressly pointed out that all such substitutions and/or modifications are intended to be encompassed by the present application. While the products and methods of preparation of the present application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the products and methods of preparation described herein without departing from the spirit and scope of the application.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The present application uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Unless specifically stated otherwise, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described herein.
In order to facilitate an understanding of the embodiments of the present application, abbreviations and key terms that may be involved in the embodiments of the present application are first explained or defined.
LC-MS/MS: liquid chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry;
cracking: a chemical process of converting a sample (mainly referred to as a high molecular compound) into another several substances (mainly referred to as low molecular compounds) by thermal energy;
and (3) reduction: a reaction for reducing the total oxidation state of carbon atoms in an organic molecule, for example, in the present application refers to the opening of disulfide bonds;
alkylation: the reaction process of introducing an alkyl group into an organic molecule by an addition or substitution reaction, for example, refers to alkylation of a mercapto group in the present application;
FNA sample: a thin needle punctures a sample;
quality control product: the application is prepared by adding a known amount of sample to be tested into biological medium for quality control, and the application has high concentration quality control (HQC) and low concentration quality control (LQC);
quality control peptide: on the basis of a peptide sequence to be detected, adding a specific number of amino acids at the N end and the C end respectively, and adopting a heavy-label amino acid at a position which can be replaced by a heavy-label isotope at the first beginning of the N end of the peptide sequence to be detected;
internal standard peptide: on the basis of a peptide sequence to be detected, the last amino acid at the C end adopts a heavy standard amino acid during synthesis;
cell line: the cell population propagated after the first passage of the primary cell culture is successful also refers to the cultured cells which can be continuously passed for a long time;
matrix (Matrix): all other components within a substance form, except the analyte;
assignment: a process of assigning a certain value to a certain variable, which means in the present application a process of obtaining the actual concentration or content of the corresponding peptide fragment by on-machine measurement;
LB solution: the Lysis Buffer solution contains 6M urea, 2M thiourea and 100mM ammonium bicarbonate;
TCEP: a reducing agent, tricarboxyethyl phosphine, tris (2-carboxyyl) phosphine;
IAA: the alkylating agent iodoacetamide, iodoacetamide.
In a first aspect, the present application provides a quality control for quality control of a thyroid nodule mass spectrometry detection process, comprising:
extracting a protein solution prepared by a cell line with thyroid cancer; and
a quality control peptide;
the quality control peptide is prepared by adding a specific number of amino acids to the N end and the C end of a polypeptide peptide fragment sequence, and adopting a heavy-standard amino acid at the position which is started at the N end of the polypeptide peptide fragment sequence and can be replaced by a heavy-standard isotope.
In some preferred embodiments, the peptide fragment length of the polypeptide peptide fragment is 7 to 38 amino acids, the isoelectric point pI is 3.77 to 9.99, and the hydrophobicity analysis GRAVY value is-1.66 to 1.41.
In some preferred embodiments, the sequence of the polypeptide peptide fragment comprises at least one of the polypeptide peptide fragment sequences of SEQ ID Nos. 1-41 as shown in Table 1.
In some preferred embodiments, the amino acid sequence of the quality control peptide comprises at least one quality control peptide sequence of SEQ ID Nos. 1-41 as shown in Table 1.
Table 1, quality control peptide sequences and polypeptide peptide fragment sequences
In some preferred embodiments, the quality control further comprises a diluent.
In some preferred embodiments, the diluent comprises any one of 0.05 to 0.5v% formic acid aqueous solution, 30 to 60v% acetonitrile aqueous solution, 30 to 60v% methanol aqueous solution, or LB solution.
In some preferred embodiments, the theoretical content of the quality control peptide in the quality control product is between the reference interval of the corresponding peptide fragment in the clinical sample.
In some preferred embodiments, the thyroid cancer extraction cell line comprises at least one of a CAL62 cell line, 8305C cell line, BCPAP cell line, 8505C cell line, BHT101 cell line, HTCC3 cell line, or K5 cell line.
In a partially preferred embodiment, the protein solution prepared with the thyroid cancer extraction cell line is in particular prepared at 8X 10 4 The cells were mixed with 5. Mu.L of LB solution by sonication.
In some preferred embodiments, the quality control product comprises at least one high concentration quality control product and at least one low concentration quality control product according to the content of the quality control peptide.
In some preferred embodiments, the quality control product comprises a high concentration quality control product and a low concentration quality control product according to the content of the quality control peptide.
In some preferred embodiments, the preparation process of the quality control product comprises:
1) Adding a diluent into the quality control peptide powder to dilute to obtain an intermediate solution;
2) Mixing the intermediate solution with the protein solution and the diluent according to the proportion to obtain the quality control product.
In a part of the preferred embodiments, the step 1) includes:
1.1 Adding a diluent into the quality control peptide powder for dilution to obtain peptide section mother liquor;
1.2 Adding a diluent into the peptide fragment mother liquor to dilute to obtain an intermediate liquor.
In some preferred embodiments, the quality control product is further subjected to pretreatment and three-person three-batch assignment operation to obtain an assignment range.
A second part, providing a quality control kit for quality control of thyroid nodule mass spectrometry detection process, comprising:
a first portion of the quality control; and
an internal standard peptide.
In some preferred embodiments, the internal standard peptide is synthesized by using a heavy standard amino acid at the last amino acid at the C-terminal based on the sequence of the peptide fragment to be detected.
The third part, the application also provides an application of the quality control product according to any one of the first part or the kit according to the second part in quality control of thyroid nodule mass spectrometry detection process.
In some preferred embodiments, the thyroid nodule mass spectrometry detection aims to accurately acquire polypeptide mass spectrometry data and/or polypeptide content derived from thyroid nodules.
In some preferred embodiments, the peptide fragment length of the polypeptide is 7 to 38 amino acids, the isoelectric point pI is 3.77 to 9.99, and the GRAVY value of the hydrophobicity assay is-1.66 to 1.41.
In some preferred embodiments, the use comprises:
s1, respectively taking a high-concentration quality control product and a low-concentration quality control product, respectively sequentially carrying out pretreatment, adding an internal standard peptide and three-person three-batch LC-MS/MS detection to obtain a high-concentration quality control product peptide fragment assignment detection content and a low-concentration quality control product peptide fragment assignment detection content, and taking a mean value +/-2 SD of each peptide fragment assignment detection content as an assignment range;
s2, performing pretreatment, adding internal standard peptide, and detecting by an LC-MS/MS method on the FNA sample with red blood cells removed to obtain mass spectrum data of each peptide segment in the FNA sample;
s3, synchronously carrying out step S2, namely respectively taking a high-concentration quality control product and a low-concentration quality control product, and respectively carrying out pretreatment, adding an internal standard peptide and detecting by an LC-MS/MS method to obtain the detection content of each peptide segment in the quality control product;
s4, comparing the detection content of each peptide segment obtained in the step S3 with the assignment range of each peptide segment obtained in the step S1, and if at least one peptide segment does not accord with the assignment result, making errors in the processing process of the FNA samples in the batch;
s5, if the detection content of each peptide segment obtained in the step S3 is within the assignment range of each peptide segment obtained in the step S1, the FNA sample pretreatment and detection process in the step S2 are correct, and a sample result is output;
in some preferred embodiments, the pretreatment, addition of internal standard peptide, and detection by LC-MS/MS method in steps S1, S2, and S3 are all the same.
In some preferred embodiments, the step of pretreatment comprises cleavage, reduction, alkylation, enzymatic hydrolysis, and desalting.
In some preferred embodiments, the internal standard peptide is synthesized by using a heavy standard amino acid at the last amino acid at the C-terminal based on the sequence of the peptide fragment to be detected.
In some preferred embodiments, the sample loading of the internal standard peptide is 10 to 5000fmol.
In some preferred embodiments, the content of the quality control peptide in the high-concentration quality control product and the low-concentration quality control product is between the reference interval of the corresponding peptide fragment in the FNA sample.
In a part of the preferred embodiments, the following steps are performed on the FNA sample in step S2 to remove red blood cells: the FNA sample is a fine needle puncture sample, and is obtained by repeatedly sucking and puncturing by using 19-27 g of syringe needle in ultrasonic guidance or operation; firstly, carrying out low-temperature 4 ℃ pyrolysis through 0.5mL of erythrocyte lysate, putting into a centrifuge after reacting for 5min, and centrifuging for 10min at 300 g; after centrifugation, the solution was discarded and the cells remaining after centrifugation were retained.
In some preferred embodiments, the pretreatment operations in steps S1, S2, S3 include cleavage, reduction, alkylation, enzymolysis, desalting, specifically: adding a lysate (6M urea, 2M thiourea), reducing agents TCEP and IAA to react in a PCT tube, and setting instrument parameters during the reaction: 90cycles, each cycle comprising 45000psi,30s, and 10s off-time; after the reaction is finished, adding 0.75-1.5 mug LysC and 2.5-5 mug Trypsin to accelerate the reaction in PCT, wherein the reaction conditions are as follows: 120cycles, each cycle comprising 20000psi,50s, and 10s off-time. After digestion, the polypeptides were desalted by a C18 column.
The fourth part, the application also provides a high-quality control thyroid nodule mass spectrometry detection method, which comprises the following steps:
s1, respectively taking a high-concentration quality control product and a low-concentration quality control product, respectively sequentially carrying out pretreatment, adding an internal standard peptide and three-person three-batch LC-MS/MS detection to obtain a high-concentration quality control product peptide fragment assignment detection content and a low-concentration quality control product peptide fragment assignment detection content, and taking a mean value +/-2 SD of each peptide fragment assignment detection content as an assignment range;
s2, performing pretreatment, adding internal standard peptide, and detecting by an LC-MS/MS method on the FNA sample with red blood cells removed to obtain mass spectrum data of each peptide segment in the FNA sample;
s3, synchronously carrying out step S2, namely respectively taking a high-concentration quality control product and a low-concentration quality control product, and respectively carrying out pretreatment, adding an internal standard peptide and detecting by an LC-MS/MS method to obtain the detection content of each peptide segment in the quality control product;
s4, if the detection content of each peptide segment obtained in the step S3 is within the assignment range of each peptide segment obtained in the step S1, the FNA sample pretreatment and detection process in the step S2 are correct, and a sample result is output;
the pretreatment, the addition of internal standard peptide and the detection operation steps of the LC-MS/MS method in the steps S1, S2 and S3 are the same.
In some preferred embodiments, the step of pretreatment comprises cleavage, reduction, alkylation, enzymatic hydrolysis, and desalting.
In some preferred embodiments, the content of the quality control peptide in the high-concentration quality control product and the low-concentration quality control product is between the reference interval of the corresponding peptide fragment in the FNA sample.
In a part of the preferred embodiments, the following steps are performed on the FNA sample in step S2 to remove red blood cells: the FNA sample is a fine needle puncture sample, and is obtained by repeatedly sucking and puncturing by using 19-27 g of syringe needle in ultrasonic guidance or operation; firstly, carrying out low-temperature 4 ℃ pyrolysis through 0.5mL of erythrocyte lysate, putting into a centrifuge after reacting for 5min, and centrifuging for 10min at 300 g; after centrifugation, the solution was discarded and the cells remaining after centrifugation were retained.
In some preferred embodiments, the pretreatment operations in steps S1, S2, S3 include cleavage, reduction, alkylation, enzymolysis, desalting, specifically: adding a lysate (6M urea, 2M thiourea), reducing agents TCEP and IAA to react in a PCT tube, and setting instrument parameters during the reaction: 90cycles, each cycle comprising 45000psi,30s, and 10s off-time; after the reaction is finished, adding 0.75-1.5 mug LysC and 2.5-5 mug Trypsin to accelerate the reaction in PCT, wherein the reaction conditions are as follows: 120cycles, each cycle comprising 20000psi,50s, and 10s off-time. After digestion, the polypeptides were desalted by a C18 column.
In some preferred embodiments, the sample results include polypeptide mass spectral data and/or polypeptide content derived from thyroid nodules.
The present application is described in detail below.
Example 1:
as shown in fig. 1, a quality control product is provided, which includes:
extracting a protein solution prepared by a cell line with thyroid cancer;
a quality control peptide; and
a dilution liquid;
the quality control peptide is prepared by adding a specific number of amino acids to the N end and the C end of a polypeptide peptide fragment sequence, and adopting a heavy-standard amino acid at the position which is started at the N end of the polypeptide peptide fragment sequence and can be replaced by a heavy-standard isotope.
The preparation process of the quality control product specifically comprises the following steps:
1) Collecting at least 1000 clinical samples, and detecting to obtain a reference interval of each peptide shown in table 2;
2) Taking part of quality control peptide powder shown in table 1, and adding diluent to obtain a series of peptide mother liquor, as shown in table 3;
3) As shown in table 3, the mother liquor of each peptide is diluted according to different proportions to obtain intermediate liquor;
4) As shown in table 3, different intermediate liquid volumes of each peptide segment are respectively mixed with protein solution and diluent to obtain low-concentration quality control product LQC and high-concentration quality control product HQC; the theoretical concentration of the quality control product is shown in table 4, and the quality control product with high and low concentration can cover a reference region of a clinical sample;
5) And the quality control product is subjected to assignment operation by using the internal standard peptide when in application.
Wherein the protein solution is specifically 4×10 4 cell 8305C cell line, 4X 10 4 The cells 8505C cell line was mixed with 5. Mu.L of LB solution and sonicated at 40KHz for 10 min. The protein solution can be prepared by mixing at least one cell line selected from a CAL62 cell line, a 8305C cell line, a BCPAP cell line, a 8505C cell line, a BHT101 cell line, a HTCC3 cell line or a K5 cell line with LB solution through ultrasound, and the types of the cell lines do not influence the quality control effect of the quality control product.
The diluent is specifically 0.1v% formic acid in water. The diluent can be any one of 0.05-0.5v% formic acid aqueous solution, 30-60v% acetonitrile aqueous solution, 30-60v% methanol aqueous solution or LB solution, and the diluent does not influence the quality control effect of the quality control product.
TABLE 2 FNA reference interval of clinical samples
TABLE 3 preparation of quality control products
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TABLE 4 theoretical amount of each peptide fragment in quality control product
Example 2:
based on the foregoing embodiments, the quality control product is applied to quality control of a thyroid nodule mass spectrometry detection process, that is, a thyroid nodule mass spectrometry detection method is provided, and first, the sample source inclusion criteria include: age 18-70 years; patients with thyroid nodule primary treatment without drug treatment; thyroid nodule is more than or equal to 5mm, thyroid fine needle is punctured, bethesda III/IV; a transthyretin total/partial resection with histological reporting of the corresponding cytopathological penetration nodule; the patients voluntarily participated in the study after informed consent. Sample source exclusion criteria included: the sample size is insufficient for the patients without operation.
Specific parameters detected by the LC-MS/MS method comprise a liquid phase module and a mass spectrum module.
1. Liquid phase module:
1) Mobile phase: the mobile phase A is 0.05-0.5% formic acid aqueous solution, and the mobile phase B is 0.05-0.5% formic acid acetonitrile solution;
2) Chromatographic column: a C18 chromatographic column;
3) Column temperature: 40 ℃;
4) Flow rate: 0.2mL/min;
5) Injector temperature: 7 ℃;
6) The elution gradient is shown in table 5.
TABLE 5 elution gradient
| Time (min) | 0.00 | 0.50 | 8.50 | 8.60 | 10.00 | 10.10 | 12 |
| ％B | 12 | 12 | 40 | 80 | 80 | 12 | 12 |
2. Mass spectrometry module:
1) The mass spectrum conditions are shown in table 6.
Table 6, mass spectrum conditions
| Ionization mode | ESI(+) |
| Scan Mode | Scheduled MRM |
| Curtain Gas(CUR) | 10 |
| Collision Gas(CAD) | 9 |
| Temperature(TEM) | 400 |
2) The mass spectral parameters are shown in table 7.
Table 7, mass spectral parameters
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The thyroid nodule mass spectrometry detection method is shown in fig. 2, and specifically comprises the following steps:
(1) respectively taking a high-concentration quality control product and a low-concentration quality control product, and respectively sequentially carrying out pretreatment: adding a lysate (6M urea, 2M thiourea), reducing agents TCEP and IAA to react in a PCT tube, and setting instrument parameters during the reaction: 90cycles, each cycle comprising 45000psi,30s, and 10s off-time; after the reaction is finished, adding 0.75-1.5 mug LysC and 2.5-5 mug Trypsin to accelerate the reaction in PCT, wherein the reaction conditions are as follows: 120cycles, each cycle comprising 20000psi,50s, and 10s off-time; desalting the polypeptide by a C18 column after digestion is finished to obtain pure polypeptide; adding the internal standard peptide shown in Table 9, detecting by an LC-MS/MS method on three batches of three persons to obtain a high-concentration quality control product peptide fragment assignment detection content and a low-concentration quality control product peptide fragment assignment detection content, and taking the average value of each peptide fragment assignment detection content + -2 SD as an assignment range; as shown in table 8;
(2) the FNA sample is a fine needle puncture sample, and is obtained by repeatedly sucking and puncturing by using 19-27 g of syringe needle in ultrasonic guidance or operation; firstly, carrying out low-temperature 4 ℃ pyrolysis through 0.5mL of erythrocyte lysate, putting into a centrifuge after reacting for 5min, and centrifuging for 10min at 300 g; after centrifugation, the solution is discarded, and the cells remaining after centrifugation are retained;
(3) removing an FNA sample of the red blood cells, performing pretreatment in the same step as the step (1), adding an internal standard peptide, and detecting by an LC-MS/MS method to obtain mass spectrum data of each peptide segment in the FNA sample;
(4) synchronously carrying out the step (3), respectively taking quality control products HQC and LQC shown in Table 3, respectively carrying out pretreatment, adding internal standard peptide and detecting by an LC-MS/MS method in the same step as the step (1), and obtaining the detection content of each peptide segment in the quality control product;
(5) comparing the detected content of each peptide fragment obtained in the step (4) with the assigned range of each peptide fragment obtained in the step (1), if the detected content of each peptide fragment is within the assigned range, performing pretreatment and detection on the FNA sample without errors, calculating according to mass spectrum data to obtain the content of each peptide fragment to be detected, and outputting partial results as shown in Table 10.
As an expansion, the obtained FNA sample mass spectrum result (mass spectrum data or the corresponding content of each peptide fragment) is substituted into a judging model, the judging model can take the mass spectrum data or the content of each peptide fragment as input, a scoring result is output, the benign and malignant of the sample can be judged according to the scoring result, the judging accuracy of the benign and malignant of the thyroid nodule sample is 100% through comparison with a clinical diagnosis result, and the result consistency information is shown in table 11.
Table 8, quality control assignment results
TABLE 9 internal standard peptide dosage
| Peptide fragment numbering | Internal standardPeptide (fmol) |
| 1 | 100 |
| 4 | 500 |
| 5 | 100 |
| 6 | 600 |
| 11 | 1500 |
| 13 | 200 |
| 15 | 500 |
| 16 | 50 |
| 20 | 800 |
| 21 | 600 |
| 23 | 200 |
| 24 | 100 |
| 29 | 400 |
| 32 | 100 |
| 38 | 300 |
TABLE 10 Mass Spectrometry results for different FNA samples (peptide fragment content, fmol)
TABLE 11 FNA sample result information
As can be seen from the results of the table 10 and the table 11, according to the scheme of the application, the FNA sample and the high-concentration quality control product are respectively preprocessed, the quality control product content is obtained after the on-machine detection, the quality control product content is compared and analyzed with the assigned result obtained after the on-machine detection is carried out after the pretreatment of the quality control product content is the same as that of the quality control product alone, the quality control of the detection process can be carried out, when the quality control product result accords with the assigned result, the quality degree of the detection process is higher, thus the error of the detection process of the thyroid nodule is smaller, the accurate mass spectrum data of the corresponding peptide segment to be detected can be obtained, the content of the corresponding peptide segment to be detected can be calculated according to the mass spectrum data, the evaluation of the malignancy degree or probability of the thyroid nodule can be completed based on the correct mass spectrum data or the peptide segment content, and the evaluation result of the thyroid nodule obtained by applying the scheme of the application and the clinical pathology detection result are completely consistent, the accuracy is 100%, the detection process is standard, and the scheme has good application prospect.
Example 3:
based on the foregoing examples, the prepared quality control product was stored at-20 ℃ for 1 month, treated and tested according to the procedure described above, 6 batches were measured, each batch was 4-fold, and the relative deviation from the assigned result (-25% stable) was calculated for 1 month of storage, and the results are shown in table 12.
Table 12, assignment result relative deviation (%)
| Peptide fragment numbering | 1 | 4 | 5 | 6 | 11 | 13 | 15 | 16 | 20 | 21 | 23 | 24 | 29 | 32 | 38 |
| HQC | -1 | 15 | 10 | 10 | 11 | -8 | 3 | 5 | -3 | 13 | 6 | 2 | 4 | 6 | -20 |
| LQC | -17 | -1 | -1 | 0 | 0 | -21 | 0 | -2 | 5 | 11 | 9 | -16 | -5 | 4 | -24 |
As can be seen from Table 12, the quality control product obtained by the application not only reduces the matrix effect after adding the protein solution, but also solves the adsorption problem of the quality control peptide, ensures the stability and accuracy of the quality control product, can be used as a quality control kit component preparation kit for quality control in the detection process of thyroid nodule mass spectrometry, and can effectively control and improve the quality control level of the mass spectrometry detection process.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the application. Various modifications or additions to the described embodiments may be made by those skilled in the art to which the application pertains or may be substituted in a similar manner without departing from the spirit of the application or beyond the scope of the appended claims.
While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and subcombinations are intended to fall within the scope of this disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. While the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the application extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Therefore, the present application is not intended to be limited by the specific disclosure of the preferred embodiments herein.
The application is a well-known technique.
Claims (10)
1. A quality control article for quality control of thyroid nodule mass spectrometry detection process, characterized in that: comprising the following steps:
extracting a protein solution prepared by a cell line with thyroid cancer; and
a quality control peptide;
the quality control peptide is prepared by adding a specific number of amino acids to the N end and the C end of a polypeptide peptide fragment sequence, and adopting a heavy-standard amino acid at the position which is started at the N end of the polypeptide peptide fragment sequence and can be replaced by a heavy-standard isotope.
2. The quality control article of claim 1, wherein:
the peptide length of the polypeptide peptide is 7-38 amino acids, the isoelectric point pI is 3.77-9.99, and the hydrophobicity analysis GRAVY value is-1.66-1.41.
3. The quality control article according to claim 1 or 2, characterized in that:
the theoretical content of the quality control peptide in the quality control product is between the reference interval of the corresponding peptide segment in the clinical sample.
4. The quality control article according to claim 1 or 2, characterized in that:
the thyroid cancer extraction cell line comprises at least one of a CAL62 cell line, 8305C cell line, BCPAP cell line, 8505C cell line, BHT101 cell line, HTCC3 cell line, or K5 cell line.
5. The quality control article according to claim 1 or 2, characterized in that: the quality control product also comprises diluent.
6. The quality control article of claim 5, wherein: the preparation process of the quality control product comprises the following steps:
1) Adding a diluent into the quality control peptide powder to dilute to obtain an intermediate solution;
2) Mixing the intermediate solution with the protein solution and the diluent according to the proportion to obtain the quality control product.
7. Use of a quality control article according to any one of claims 1 to 6 in quality control of thyroid nodule mass spectrometry detection procedures.
8. The use according to claim 7, characterized in that
The thyroid nodule mass spectrometry detection aims to accurately acquire polypeptide mass spectrometry data and/or polypeptide content derived from thyroid nodules.
9. The use according to claim 8, characterized in that:
the peptide length of the polypeptide is 7-38 amino acids, the isoelectric point pI is 3.77-9.99, and the hydrophobicity analysis GRAVY value is-1.66-1.41.
10. A thyroid nodule mass spectrometry detection method with high quality control is characterized in that: comprising the following steps:
s1, respectively taking a high-concentration quality control product and a low-concentration quality control product, respectively sequentially carrying out pretreatment, adding an internal standard peptide and three-person three-batch LC-MS/MS detection to obtain a high-concentration quality control product peptide fragment assignment detection content and a low-concentration quality control product peptide fragment assignment detection content, and taking a mean value +/-2 SD of each peptide fragment assignment detection content as an assignment range;
s2, performing pretreatment, adding internal standard peptide, and detecting by an LC-MS/MS method on the FNA sample with red blood cells removed to obtain mass spectrum data of each peptide segment in the FNA sample;
s3, synchronously carrying out step S2, namely respectively taking a high-concentration quality control product and a low-concentration quality control product, and respectively carrying out pretreatment, adding an internal standard peptide and detecting by an LC-MS/MS method to obtain the detection content of each peptide segment in the quality control product;
s4, if the detection content of each peptide segment obtained in the step S3 is within the assignment range of each peptide segment obtained in the step S1, the FNA sample pretreatment and detection process in the step S2 are correct, and a sample result is output;
the pretreatment, the addition of internal standard peptide and the detection operation steps of the upper LC-MS/MS method in the steps S1, S2 and S3 are the same.
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| CN115436640A (en) * | 2022-11-07 | 2022-12-06 | 西湖欧米(杭州)生物科技有限公司 | Surrogate matrix for polypeptides that can assess the malignancy or probability of thyroid nodules |
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