CN112924689B - Diabetes diagnosis kit based on quantitative determination of polypeptide combined marker and method thereof - Google Patents
Diabetes diagnosis kit based on quantitative determination of polypeptide combined marker and method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- 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
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Abstract
The invention relates to the field of medical biological detection, in particular to a new application of a polypeptide combined marker in preparing a diabetes diagnosis reagent or a kit. The invention provides a method and a kit for quantitatively detecting endogenous polypeptides in serum by utilizing liquid chromatography-mass spectrometry Multiple Reaction Monitoring (MRM). The reagent and the method can quantitatively determine the content of endogenous 4 polypeptide markers in human serum, and further calculate the variable of the combined marker and the determined intercept value based on a binary logistic regression equation, and are used for early warning and/or diagnosis of diabetes. The kit and the detection method have the advantages of good stability, high specificity and potential for clinical popularization.
Description
Technical Field
The invention relates to the field of medical biological detection technology and diagnosis, prevention and treatment of metabolic diseases, in particular to a method for quantitatively determining human plasma endogenous polypeptide and a kit and a method for preparing early warning and/or diagnosis of diabetes by using a polypeptide combined marker.
Background
According to 2017, international diabetes union data show that there are 4.15 hundred million diabetics all over the world, wherein type 2 diabetes (T2 DM) accounts for more than 95%. The global diabetes population is expected to increase to 5.52 hundred million by 2030. Diabetes mellitus is the 3 rd largest disease that endangers human health following cardiovascular diseases and tumors. At present, although the diagnostic standard of type 2 diabetes is clear and has gold standards such as Oral Glucose Tolerance Test (OGTT), the diabetes has no obvious symptoms in the initial stage and has long latent period, so that the disease condition is irreversible and the optimal intervention period is missed when the symptoms appear or the intervention treatment is carried out after clinical diagnosis is confirmed, and the finding of the biomarker for early warning and diagnosis has important significance for researching the pathology, classification, early diagnosis, delaying and preventing the occurrence of the diabetes.
The discovery of new diagnostic markers using multigroup chemistry techniques is a hot spot in current research. The polypeptide plays an important role in the pathogenesis and intervention treatment of diabetes, such as peptide preparations of various polypeptide drugs, such as glucagon-like peptide-1 (GLP-1) analogues, dolaglutide (dulaglutide), Brain Natriuretic Peptide (BNP) and the like. The polypeptide can embody the processes of synthesis, processing and degradation of protein under different physiological and pathological conditions, and has a regulating effect on the processes of gene expression, substance metabolism and the like, so that the polypeptide omics technology is adopted to discover the novel polypeptide biomarker for early warning/diagnosis of diabetes mellitus, and the polypeptide biomarker has great potential.
And (3) marker verification: the currently discovered type 2 diabetes potential biomarkers include gene variation, RNA transcripts, polypeptides, proteins, lipids, small molecule metabolites (branched chain amino acids/aromatic amino acids) and the like, but mostly come from complication samples, such as diabetic nephropathy, diabetes accompanied with hypertension and hyperglycemia samples, gestational diabetes and the like (Chinese patent CN 106645757A; medical reviews, 2019, (20), 4080-; in addition, after the differential markers are screened by adopting a multigroup chemical technology, further clinical sample verification work is lacked, so that the clinical application value is limited; or ELISA or Western blot verification is adopted, the experiment cost is high, and the flux is insufficient (Chinese patent CN 108957011; chromatogram, 2019, 37 (8): 853-.
Based on the problems in the research, the invention adopts a method of combining non-targeted peptidomics and targeted quantitative peptidomics, and the method has the characteristics of high flux, good data repeatability and high sensitivity. The method comprises the steps of screening potential diagnostic markers by adopting a strategy from discovery to verification, successfully screening and verifying a combined marker of endogenous polypeptides in human serum from a clinical sample (without complications) in the early stage of diabetes, wherein the combined marker can be used for early warning and/or diagnosis of diabetes, has good diagnostic sensitivity and specificity, and is used for quantitatively determining the content of target polypeptides and for early warning and/or diagnosis of diabetes, and a kit for quantitatively determining the content of the target polypeptides and for early warning and/or diagnosis of diabetes is prepared.
Disclosure of Invention
The invention aims to solve the problems of lack of early warning and diagnosis markers of diabetes, low sensitivity, low specificity and the like, adopts the strategies of discovering differential polypeptides by non-targeted proteomics and verifying targeted proteomics, provides an application and a kit of a combined polypeptide marker in early warning/diagnosis of diabetes, and also provides a method for quantitative analysis and detection of the combined polypeptide marker.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
(1) non-targeted analysis is carried out on endogenous polypeptides in serum of normal people, patients with early diabetes and patients with diabetes (without complications such as hypertension, hyperlipidemia and the like) by adopting a non-targeted proteomics method, differential polypeptides are screened by a non-standard quantitative method, screening conditions comprise a P value, a credible confidence interval and the like, and the differential polypeptides meeting the conditions are reserved.
(2) And (2) verifying the differential polypeptide in the step (1) in another batch of clinical samples of healthy people/diabetic patients (without complications such as hypertension, hyperlipidemia and the like) by synthesizing a standard peptide and a dimethyl marker and adopting a targeted proteomics method. And the stability of the polypeptide is considered, and the potential of the polypeptide for preparing and developing a diabetes early warning/diagnosis kit is preliminarily evaluated.
(3) And (3) adopting SPSS statistical software, performing binary logistic regression analysis to obtain regression as a combined marker variable, and adopting an ROC (receiver operating characteristic) curve to evaluate the sensitivity and the specificity of the combined marker.
(4) Use of combination markers: the content of polypeptides GVLSSRQLGLPGPPDVPDHAAYHPF (245), GVLSSRQLGLPGPPDVPDHAA (244), GLPGPPDVPDHAAYHPF (251), GSEMVVAGKLQ (261) varies significantly in diabetic patients. The 4 polypeptides regressed into the combined marker variable X, and the binary logistic regression equation was:
X＝1.630+5.279×C(245)+8.971×C(244)-4.232×C(251)-14.175×C(261)
prob (diabetes) 1/[1+ e-X]
Wherein C represents the concentration of plasma polypeptide; e is the Euler number, the base of the natural logarithm function; prob is the variable value of the combined marker, when the P value is more than or equal to 0.55, the patient is diagnosed as diabetic, otherwise, the patient is non-diabetic.
(5) The diagnostic system comprises means for: the chromatographic column and the detecting instrument are a liquid chromatogram-mass spectrometer.
(6) Determining the composition of the kit, wherein the kit comprises seven reagents or solutions A, B, C, D, E, F and G, and specifically comprises the following steps:
dimethyl labeling reagent: a, PBS buffer (pH 8.0-8.4); b, formaldehyde at a concentration of 4% by volume (light standard treatment agent) and/or deuterated formaldehyde at a concentration of 4% by volume (medium standard treatment agent); c,0.6M sodium cyanoborohydride (NaBH3 CN); d, ammonia water with the mass concentration of 1%; e, 5% by volume formic acid solution;
standard solution F: treating polypeptide (245, 244, 251, 261) with dimethyl light standard reagent to obtain light standard solution, drying, desalting, drying again, dissolving in formic acid with volume concentration of 0.1% -1%, and final concentration of polypeptide is 10-40 μ g/mL respectively;
internal standard solution G containing stable isotope: the polypeptide (245, 244, 251, 261) is processed by a dimethyl labeled bid-winning reagent to obtain a bid-winning solution, and the bid-winning solution is dissolved in formic acid with the volume concentration of 0.1-1% after being dried, desalted and dried again, and the final concentration of the polypeptide is 0.3-10 mug/mL respectively.
The method comprises the following specific steps:
1) and (3) ultrafiltration: taking 10-50 mu L of plasma sample, and centrifuging by a 10K Da ultrafiltration tube to extract endogenous polypeptide; drying to obtain an endogenous polypeptide sample;
2) light mark marking: sequentially treating the endogenous polypeptide sample by using the dimethyl-labeled light standard reagent A, B, C, D, E to obtain an endogenous polypeptide dimethyl-labeled sample (light standard), and drying;
3) desalting: desalting with TIP head or solid phase extraction column filled with C18, and drying; refrigerating in a refrigerator at minus 80 ℃ for standby;
4) preparing a test solution: dissolving the endogenous polypeptide light marker sample with 10-50 μ L of 0.1% -1% formic acid solution, centrifuging at 20000G for 5-8 min, collecting supernatant 10-40 μ L, and adding 10-40 μ L of internal standard solution G to obtain the sample solution to be tested;
5) preparation of a standard curve: diluting the standard solution F with 0.1-1% formic acid solution to obtain a series of standard solutions, taking 1-10 μ L of the standard solution, adding 10-40 μ L of blank plasma sample and 10-40 μ L of internal standard solution G to obtain a series of standard curve samples, wherein the final concentration range is 5ng/mL-1000 ng/mL; wherein the blank plasma sample is obtained by sequentially treating the endogenous polypeptide extracted by ultrafiltration with A, D, E, desalting, drying and dissolving in 0.1-1% formic acid solution; measuring the intensities of light standard and medium-winning ion peaks of the polypeptide by adopting liquid chromatography-mass spectrometry multiple reaction monitoring (LC-MS/MRM), and fitting a standard curve according to the comparison of the peak areas of the light standard and the medium-winning ion peaks;
6) polypeptide content determination: and respectively analyzing the prepared standard curve sample and the clinical sample test solution by adopting LC-MS/MRM, and calculating the content of the target polypeptide by an external standard method.
(7) Conditions of instrumental analysis
The operating conditions of the liquid chromatography are as follows: waters UPLC liquid chromatograph, chromatography column, Acquity UPLC @ HSS, T3,1.8 μm; mobile phase, acetonitrile-0.1% formic acid (a), 0.1% aqueous formic acid (B). The gradient conditions were as follows:
TABLE 1 liquid chromatography conditions
The mass spectrometry conditions, the Qtrap 5500 mass spectrometer, the mass spectrometry Multiple Reaction Monitoring (MRM), the MRM quantitative ion pairs of the target 4 polypeptides and the mass spectrometry parameters are shown in Table 2, and the mass spectrogram is shown in FIG. 1.
TABLE 2 quantitative ion pairs of target polypeptides
(7) Serum samples were used to test the efficacy of the invention. Normal persons can be well distinguished from diabetic patients by using the combined markers, and as shown in fig. 2, the AUC value is 0.864, the sensitivity is 0.78, and the specificity is 0.78. The optimum critical point (cut-off) was 0.55. Diabetes is diagnosed as having a value greater than or equal to the cut-off point value.
(8) Stability study of polypeptide markers: the 4 polypeptides related to the patent can be stored in a refrigerator at 4 ℃ for 2 months, and no matter light standard samples or successful bid samples are obviously degraded, which indicates that the kit has good stability.
The invention has the advantages that:
1) the discovery process of the biomarker comprises three processes of screening differential polypeptides by adopting a non-targeted metabonomics method, further confirming the structure by polypeptide synthesis and verifying clinical samples of different batches by adopting a targeted quantitative polypeptinomics method of mass spectrometry Multiple Reaction Monitoring (MRM), and has higher clinical reference value and reliability.
2) The invention provides a kit for early warning/diagnosis of diabetes, which can be used for conveniently realizing accurate quantitative determination of target polypeptide in a clinical sample to be detected, further calculating a combined marker variable Prob and a judgment cut-off value (cut-off) based on a binary logistic regression equation, and making preliminary judgment on the risk and diagnosis of the diabetes of the sample to be detected. The kit has the characteristics of good stability, high diagnosis specificity and high sensitivity, is applied to auxiliary early diagnosis, and has higher development and application values.
Drawings
FIG. 1 MRM detection map of the polypeptide
FIG. 2 ROC diagnostic model; drawing notes: area under the AUC curve; sensitivity, specificity, criterion cut-off cutoff
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention. These examples are illustrative only and do not limit the scope of the present invention in any way. Modifications and substitutions in detail and form may be made to the technical solution of the present invention without departing from the spirit and scope of the present invention, but the modifications and substitutions are within the scope of the present invention.
Example 1: screening of differential polypeptides by non-standard quantitative method
Clinical samples: 23 human serum samples (including 7 healthy person serum samples, 7 pre-diabetic patient serum samples, and 9 type 2 diabetic patient serum samples) were obtained from the sixth national hospital of Shanghai. The patients with pre-diabetes and type 2 diabetes are diagnosed or excluded by experts, and the age, Body Mass Index (BMI), blood sugar and other indexes of the three groups of tested people are matched. Blood samples were approved by the local hospital ethics committee and all subjects signed informed consent. Blood samples were collected and stored in a-80 ℃ freezer.
Extracting endogenous polypeptide in serum: an ultrafiltration centrifugal method is adopted. The specific operation flow is as follows: blood samples were taken from-80 ℃ freezer and thawed in ice bath, vortexed, mixed, centrifuged at 20000g for 5min and lipid discarded. mu.L of each serum sample was taken, 125. mu.L of deionized water was added and the mixture was boiled for 5min for denaturation. After cooling to room temperature, 150 μ L of an aqueous solution of 40% acetonitrile-0.1% formic acid by volume was added, vortexed, mixed well and allowed to stand in an ice bath for 40 min. The sample was then transferred to an ultrafiltration tube with a molecular weight cut-off of 10kDa, centrifuged at 6000g at 4 ℃ for 20min and the filter cake was washed twice with 300. mu.L of an aqueous solution of 20% acetonitrile-0.1% formic acid by volume. Collecting filtrate, freeze drying, desalting, and storing in-80 deg.C refrigerator for use. Before sample injection analysis, the sample is redissolved in 25 mu L FA solution with volume concentration of 0.1%, vortexed and mixed uniformly for 2min, 20000g, centrifuged at 4 ℃ for 5min, and the supernatant is taken for nano-LC-MS/MS analysis.
LC-MS/MS data acquisition: a Nano-RPLC-MS/MS system comprising high performance liquid chromatography with quaternary gradient pump (Ultimate 3000), autosampler and linear ion trap-static orbital trap (LTQ-Orbitrap), trap column (3cm × 200 μm, C18); analytical column (12 cm. times.150. mu.m, C18). Mobile phase composition: the phase A is 0.1% formic acid aqueous solution, and the phase B is 80% acetonitrile containing 0.1% formic acid solution. The separation gradient was set as: 0-2% B for 10 min; 2-5% of B, 3 min; 5-28% of B, 60 min; 28-45% of B, 15 min; 45-95% B for 1 min; 95% B, 5 min. The flow rate was 0.5. mu.L/min. Mass spectrum conditions: the temperature of the LTQ ion transmission tube was set at 275 deg.C and the electrospray voltage was set at 2.7 kV. All data acquisition modes are data-dependent modes (DDA), mass spectrum full scan is acquired in Orbitrap, scan range is 200-. Secondary mass spectrometry scans were acquired in LTQ using Collision Induced Dissociation (CID) fragmentation. The Xcalibur software (version 2.1, Thermo corporation) was used for system control and data collection.
Mass spectrum data retrieval and analysis: the mass spectrometry data were subjected to data retrieval using Mascot software, and the database was retrieved as a human source library (UniProt, about 17 ten thousand proteins). The search parameters are set as: methionine residue (+15.9949Da) was variably modified, no cleavage, a parent ion mass error of 20ppm, and a secondary fragment ion error of 0.8 Da. The polypeptide screening threshold was set at a score above 20 and a false positive rate (FDR) of less than 1%. Polypeptide quantification data analysis was performed using MaxQuant software. The false positive rate (FDR) was 0.01.
Screening the differential polypeptides, wherein 163 differential polypeptides are screened. Further select 21 of the more fold-difference changes for further clinical sample validation, the difference polypeptide information is shown in table 3:
TABLE 3 Difference polypeptide information to further validate
| Polypeptide sequence | Multiple of difference | MW | No. | PI | GRAVY |
| GKSSSYSKQFTSSTSYNRGDSTFES | Adjusted up 2 times | 2738.82 | 25 | 8.43 | -1.34 |
| GVLSSRQLGLPGPPDVPDHAAYHPF | PRE is adjusted up 3 times | 2627.94 | 25 | 5.98 | -0.292 |
| SSYSKQFTSSTSYNRGDSTFES | PRE, T2D Down-regulated by a factor of 4-5 | 2466.51 | 22 | 5.79 | -1.286 |
| SYKMADEAGSEADHEGTHSTKR | PRE downregulation by a factor of 5 | 2407.51 | 22 | 5.37 | -1.582 |
| GVLSSRQLGLPGPPDVPDHAA | 2.4 times of PRE Up-regulation | 2083.33 | 21 | 5.21 | -0.19 |
| FTSSTSYNRGDSTFESKS | 3-fold downregulation of T2D | 2001.05 | 18 | 6.07 | -1.217 |
| DQEQSQVAEKPMEGESR | PRE Down-regulated by 3-fold | 1948.05 | 17 | 4.25 | -1.888 |
| GLGHGHEQQHGLGHGHKF | 2-fold Up-Regulation of PRE and 11-fold Down-Regulation of T2D | 1933.08 | 18 | 7.16 | -1.244 |
| HKSEVAHRFKDLGEEN | T2D Up-regulated by 3 times | 1896.05 | 16 | 6.03 | -1.55 |
| SSSYSKQFTSSTSYNRG | PRE Down-regulated by 3-fold | 1886.95 | 17 | 9.7 | -1.329 |
| EALHNHYTQKSLSLSPG | T2D Up-regulated by 5 times | 1882.06 | 17 | 7.01 | -0.824 |
| SSKITHRIHWESASLL | PRE is down-regulated by a factor of 2 and T2D is up-regulated by a factor of 2 | 1865.12 | 16 | 8.51 | -0.294 |
| GLPGPPDVPDHAAYHPF | PRE 3-fold up | 1786.96 | 17 | 5.05 | -0.535 |
| SKITHRIHWESASLL | T2D Down-regulated by 2.8-3.8 fold | 1778.04 | 15 | 8.51 | -0.26 |
| SEETKENEGFTVTAEG | PRE 3-fold up | 1727.76 | 16 | 3.98 | -1.238 |
| SRQLGLPGPPDVPDHAA | 3-fold downregulation of T2D | 1726.91 | 17 | 5.19 | -0.635 |
| GVEVHNAKTKPREEQ | T2D Up-regulated by 3 times | 1721.89 | 15 | 6.76 | -1.7 |
| DIQMTQSPSSVSASVGD | T2D Up-regulated by 3 times | 1708.81 | 17 | 3.56 | -0.241 |
| DEAGSEADHEGTH | PRE Down-regulated by 3-fold | 1354.27 | 13 | 4.17 | -1.738 |
| GVNDNEEGFF | T2D Up-regulated by 3 times | 1127.13 | 10 | 3.57 | -0.85 |
| GSEMVVAGKLQ | T2D Down-regulated by 5-fold | 1118.31 | 11 | 6 | 0.309 |
Note: the expression of up-regulation and down-regulation means that the ratio of the content of the polypeptide in the PRE group (initial diabetes group) or the T2D group (diabetes group) to the content of the corresponding polypeptide in the control group is up-regulation when the ratio is more than 1; down regulation is less than 1.
Example 2: dimethyl-tagged polypeptides and targeted MRM validation
Clinical samples: 18 human serum samples (including 9 serum samples from healthy subjects and 9 serum samples from type 2 diabetic patients) were obtained from the sixth national hospital of Shanghai. The patients with type 2 diabetes are diagnosed or excluded by experts, and the indexes of the age, the Body Mass Index (BMI), the blood sugar and the like of the three groups of tested people are matched. The diabetic patients have no complications such as hypertension, hyperglycemia and the like. Blood samples were approved by the local hospital ethics committee and all subjects signed informed consent. Blood samples were collected and stored in a-80 ℃ freezer.
Extracting endogenous polypeptide in serum: reference is made to example 1.
Polypeptide synthesis: based on the information in Table 3 above, the synthetic target peptides were reconfirmed by comparing their MS patterns.
Dimethyl labeling: the following kit is adopted to prepare a medium-winning internal standard solution and prepare a standard curve sample and a clinical sample test solution. The kit comprises the following components:
dimethyl labeling reagent: a, PBS buffer (PH 8.0); b, formaldehyde at a concentration of 4% by volume (light standard treatment agent) and/or deuterated formaldehyde at a concentration of 4% by volume (medium standard treatment agent); c,0.6M sodium cyanoborohydride (NaBH3 CN); d, ammonia water with the mass concentration of 1%; e, 5% by volume formic acid solution;
standard solution F: treating the polypeptide (245, 244, 251, 261) with a dimethyl light standard reagent to obtain a light standard solution, drying, desalting, drying again, dissolving in formic acid with the volume concentration of 1%, wherein the final concentration of the polypeptide is 40 μ g/mL respectively;
internal standard solution G containing stable isotope: the polypeptide (245, 244, 251, 261) is processed by a dimethyl labeled bid-winning reagent to obtain a bid-winning solution, and the bid-winning solution is dissolved in formic acid with the volume concentration of 0.1-1% after being dried, desalted and dried again, and the final concentration of the polypeptide is 10 mu g/mL respectively.
The method comprises the following specific steps:
1) and (3) ultrafiltration: taking 25 mu L of plasma sample, and centrifuging by a 10K Da ultrafiltration tube to extract endogenous polypeptide; drying to obtain an endogenous polypeptide sample;
2) light mark marking: sequentially treating the endogenous polypeptide sample by using the dimethyl-labeled light standard reagent A, B, C, D, E to obtain an endogenous polypeptide dimethyl-labeled sample (light standard), and drying;
3) desalting: desalting with TIP head or solid phase extraction column filled with C18, and drying; refrigerating at-80 deg.C in refrigerator;
4) preparing a test solution: back dissolving the endogenous polypeptide light label sample with 30 μ L of 1% formic acid solution, centrifuging for 8 minutes at 20000G, collecting supernatant 25 μ L, and adding 10 μ L of internal standard solution G to obtain the test solution to be tested;
5) preparation of a standard curve: diluting the standard substance solution F with 1% formic acid solution to obtain a series of standard substance solutions, taking 5 μ L of the standard substance solution, adding 40 μ L of blank plasma sample and 10 μ L of internal standard solution G to obtain a series of standard curve samples, wherein the final concentration ranges are 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL and 1000 ng/mL; wherein the blank plasma sample is obtained by sequentially treating the endogenous polypeptide extracted by ultrafiltration with A, D, E, desalting, drying and dissolving in 1% formic acid solution; measuring the intensities of light standard and medium-winning ion peaks of the polypeptide by adopting liquid chromatography-mass spectrometry multiple reaction monitoring (LC-MS/MRM), and fitting a standard curve according to the comparison of the peak areas of the light standard and the medium-winning ion peaks;
6) polypeptide content determination: and respectively analyzing the prepared standard curve sample and the clinical sample test solution by adopting LC-MS/MRM, and calculating the content of the target polypeptide by an external standard method.
Conditions of instrumental analysis
The operating conditions of the liquid chromatography are as follows: waters UPLC liquid chromatograph, chromatography column, Acquity UPLC @ HSS, T3,1.8 μm; mobile phase, acetonitrile-0.1% formic acid (a), 0.1% aqueous formic acid (B). Gradient conditions are shown in table 1.
The mass spectrometry conditions, Qtrap 5500 mass spectrometer, mass spectrometry Multiple Reaction Monitoring (MRM), and the MRM qualitative and quantitative ion pairs and mass spectrometry parameters of the target 21 polypeptides are shown in table 4.
Qualitative and quantitative ion pairs of table 421 polypeptides
Note: l represents a light bid and M represents a medium bid.
Target verification: the quantitative result shows that 4 polypeptides in the polypeptide library have obvious difference in normal human and diabetes samples, and can be used for a variable regression diagnosis model. Thereby further optimizing the quantitative ion pairs for 4 polypeptides, as shown in table 2.
Evaluation of stability: the stability of the target polypeptide is further evaluated, and the result shows that 4 polypeptides are placed in a refrigerator at 4 ℃ for 2 months, are not obviously degraded, and have the potential for diagnosis markers.
Example 3: determination of early warning/diagnostic markers
From the above quantitative results, it was found that the contents of the polypeptides GVLSSRQLGLPGPPDVPDHAAYHPF (245), GVLSSRQLGLPGPPDVPDHAA (244), GLPGPPDVPDHAAYHPF (251) and GSEMVVAGKLQ (261) were significantly changed in both the samples of healthy persons and diabetic patients.
The sensitivity and specificity of the combined marker are evaluated by adopting SPSS statistical software and a ROC (receiver operating characteristic) curve through regression analysis of 4 polypeptides into the combined marker variable by binary logistic regression analysis. And determining the candidate marker by the AUC of more than 0.8.
The 4 polypeptides regressed into the combined marker variable X, and the binary logistic regression equation was:
X＝1.630+5.279×C(245)+8.971×C(244)-4.232×C(251)-14.175×C(261)
prob (diabetes) 1/[1+ e-X]
Wherein C represents the concentration of plasma polypeptide; e is the Euler number, the base of the natural logarithm function; prob is the variable value of the combined marker, when the P value is more than or equal to 0.55, the patient is diagnosed as diabetic, otherwise, the patient is non-diabetic.
The cutoff value is determined by working characteristic curve (ROC curve) of the testee according to the P value of the combined marker variable, and taking the P value with the maximum sum of sensitivity and specificity as the optimal cutoff value.
Serum samples were used to test the efficacy of the invention. Normal persons can be well distinguished from diabetic patients by using the combined markers, and as shown in fig. 2, the AUC value is 0.864, the sensitivity is 0.78, and the specificity is 0.78. The diagnosis model of the invention has better clinical application effect.
Claims (4)
1. Application of the combined marker of the polypeptides GVLSSRQLGLPGPPDVPDHAAYHPF 245, GVLSSRQLGLPGPPDVPDHAA 244, GLPGPPDVPDHAAYHPF 251 and GSEMVVAGKLQ 261 in preparation of a diabetes early warning and/or diagnosis kit;
the diagnostic kit detects the content of the polypeptides 245, 244, 251 and 261 in serum.
2. Use of a combination marker according to claim 1 for the preparation of a kit for diagnosing a diabetic patient in a subject, wherein the diagnostic kit is a combination of reagents for detecting the amount of the polypeptides 245, 244, 251 and 261 using liquid chromatography-mass spectrometry.
3. A test kit for early warning/diagnosis of diabetes, wherein the diagnosis or kit comprises:
(1) dimethyl labeling reagent: a, PBS buffer solution pH = 8.0-8.4; b, a formaldehyde light standard treatment reagent with the volume concentration of 4% and/or a standard treatment reagent with the volume concentration of 4% deuterated formaldehyde; c,0.6M sodium cyanoborohydride (NaBH3 CN); d, ammonia water with the mass concentration of 1%; e, 5% formic acid solution by volume;
(2) standard solution F: treating the polypeptides 245, 244, 251 and 261 with a dimethyl light standard reagent to obtain a light standard solution, drying, desalting and drying again, dissolving the light standard solution in formic acid with the volume concentration of 0.1-1%, wherein the final concentration of the polypeptides is 10-40 mug/mL respectively;
(3) internal standard solution G containing stable isotope: the polypeptides 245, 244, 251 and 261 are processed by a dimethyl labeled bid-winning reagent to obtain a bid-winning solution, and the bid-winning solution is dissolved in formic acid with the volume concentration of 0.1-1% after being dried, desalted and dried again, and the final concentration of the polypeptides is 0.3-10 mug/mL respectively.
4. Use of a combination marker according to claim 1 for the preparation of a kit for diagnosing a diabetic patient in a subject, wherein the combination marker variables and cut-off values are used for early diagnosis and warning of diabetes; the content of the endogenous polypeptide marker in human serum can be quantitatively determined, and then the combined marker variable and the determined intercept value are calculated based on a binary logistic regression equation for early warning and/or diagnosis of diabetes.
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