A polypeptide and its use in preparing an ICP-assisted diagnosis kit
By screening for ICP-specific small molecule peptides QGAKIPKPEAS and preparing an ICP-aided diagnostic kit, the problem of insufficient sensitivity and specificity in ICP diagnosis in existing technologies has been solved, enabling early diagnosis and prediction and reducing the risk of maternal and infant complications.
Patent Information
- Application Number
- CN202310044265.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-01-30
- Publication Date
- 2026-01-06
- Estimated Expiration
- 2043-01-30
AI Technical Summary
Current technologies for diagnosing ICP mainly rely on total bile acid levels, which have low sensitivity and specificity and cannot meet the needs of early diagnosis and prediction. There is also a lack of stable serum small molecule peptide markers for auxiliary diagnosis.
The small molecule peptide QGAKIPKPEAS, which is specific to ICP, was screened out as a diagnostic biomarker. An ICP-assisted diagnostic kit was prepared using MRM technology, and the kit was then detected using TMT labeling and LC-MS/MS mass spectrometry.
It improves the diagnostic sensitivity and specificity of ICP, provides laboratory support for early diagnosis and prediction, reduces the risk of maternal and infant complications, simplifies the diagnostic process, and provides a basis for clinical intervention.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of peptidomics and reproductive medicine, and relates to a polypeptide and its application in the preparation of ICP-assisted diagnostic kits. Background Technology
[0002] Intrahepatic cholestasis of pregnancy (ICP) is a relatively common complication of pregnancy, with unexplained pruritus as its main characteristic symptom. Laboratory indicators show abnormally elevated serum total bile acid levels, often accompanied by abnormal liver enzyme levels. ICP has serious adverse effects on the fetus, leading to fetal distress, spontaneous preterm birth, and even stillbirth, increasing perinatal morbidity and mortality. Studies have indicated that the fetal mortality rate due to ICP is approximately 6-10 times higher than in normal pregnancies, making early diagnosis and intervention extremely important and urgent. However, current clinical diagnosis of ICP mainly relies on elevated total bile acid levels, which has low sensitivity and specificity, and is insufficient for early diagnosis and prediction of the disease. Therefore, it is urgent to explore the early sensitive molecular events in the occurrence of ICP and screen for susceptible biomarkers. This can provide an effective means for the diagnosis and intervention of ICP, reduce maternal and infant complications caused by the disease, and alleviate the disease pain and economic pressure on mothers, infants and their families. It has great scientific significance for promoting maternal and infant health.
[0003] Peptidomics is an emerging branch of proteomics that primarily studies endogenously produced protein fragments, also known as polypeptides or small peptides. Small peptides, composed of amino acid sequences with a molecular weight less than 10 kDa, are considered protein metabolites that can reflect the physiological state of an organism. They possess specific functions in regulating and guiding biochemical reactions, and can also assess protease activity, degradation, and denaturation. Recent studies have found that peptidomics is an effective means of discovering new biomarkers. Small peptides in tissues or body fluids can provide timely diagnostic information for diseases, becoming a highly anticipated omics platform for screening sensitive predictive biomarkers. The application of peptidomics in the diagnosis and pathogenesis research of pregnancy-related diseases has become a popular emerging field. Peptidomics can reveal the pathogenesis of ICP from a completely new perspective and identify sensitive molecular diagnostic biomarkers.
[0004] However, there are currently no reports on relatively stable serum small molecule peptide markers for the auxiliary diagnosis of ICP. If small molecule peptides that are specifically or abnormally expressed in ICP can be screened as molecular markers and corresponding auxiliary diagnostic kits can be developed, it will greatly improve the current status of ICP diagnosis in my country. Summary of the Invention
[0005] The primary objective of this invention is to address the aforementioned technical problems by providing a small molecule peptide biomarker in pregnant serum / plasma related to ICP-assisted diagnosis.
[0006] The second objective of this invention is to provide an application of small molecule peptide biomarkers from gestational serum / plasma for ICP-assisted diagnosis based on mass spectrometry multiple reaction monitoring (MRM) technology.
[0007] A third objective of this invention is to provide a kit for ICP-assisted diagnosis.
[0008] The objective of this invention is achieved through the following technical solutions:
[0009] A polypeptide for preparing an auxiliary diagnostic kit for intrahepatic cholestasis of pregnancy, with the amino acid sequence: QGAKIPKPEAS (SEQ ID NO.1).
[0010] Application of QGAKIPKPEAS (SEQ ID NO.1) as a detection target in the preparation of an auxiliary diagnostic kit for intrahepatic cholestasis of pregnancy.
[0011] Application of QGAKIPKPEAS detection reagents in the preparation of an auxiliary diagnostic kit for intrahepatic cholestasis of pregnancy.
[0012] A diagnostic kit for intrahepatic cholestasis of pregnancy, comprising reagents and consumables for detecting QGAKIPKPEAS.
[0013] As a preferred embodiment of the present invention, the kit includes reagents and consumables for detecting QGAKIPKPEAS using MRM technology.
[0014] As a preferred embodiment of the present invention, the diagnostic kit contains the target peptide of QGAKIPKPEAS, a chromatographic column (BEH-C18 column, 1.7 μm, 2.1 × 100 mm), urea, acetic acid, double-distilled water, formic acid, acetonitrile, methanol, dithiothreitol, iodoacetamide, trifluoroacetic acid, and a protease inhibitor.
[0015] The invention lies in the discovery that QGAKIPKPEAS can serve as a serum / plasma small molecule peptide biomarker for ICP-assisted diagnosis.
[0016] Specifically, QGAKIPKPEAS was screened as a serum / plasma small molecule peptide marker through the following methods: (1) Establishing a standardized specimen bank and database: Blood samples meeting the standards were collected according to standard operating procedures (SOPs), and complete demographic and clinical data were systematically collected. (2) Serum peptidomics analysis of ICP patients: ICP cases and healthy female controls of the same age as ICP cases were selected. The content of each small molecule peptide in the serum of ICP cases and control groups was detected by TMT labeling and LC-MS / MS mass spectrometry. The commonalities and characteristics of small molecule peptides between ICP cases and healthy female controls were analyzed, and differentially expressed small molecule peptides were screened. (3) Screening for disease-specific serum / plasma small molecule peptides: The screened differentially expressed small molecule peptides in serum / plasma were quantitatively analyzed in a large sample population to determine ICP-specific serum / plasma small molecule peptides.
[0017] Beneficial effects:
[0018] The advantages of serum / plasma QGAKIPKPEAS provided by this invention as a biomarker for ICP diagnosis are as follows:
[0019] This invention collects compliant blood samples using standard operating procedures (SOPs) and systematically gathers complete demographic and clinical data (which can be used to assess disease progression and the impact of factors such as patient age on the onset of the disease). Employing a peptidomics approach based on TMT labeling and LC-MS / MS mass spectrometry, the study investigated changes in small molecule peptides in the serum of primiparous, singleton pregnancies with ICP and age-matched healthy pregnant women. The results revealed that QGAKIPKPEAS is a highly specific and sensitive small molecule peptide highly correlated with the pathogenesis of ICP. Multivariate logistic regression analysis showed a significant association between QGAKIPKPEAS and the onset of ICP, with an ROC (as shown in the attached figure) and an AUC of 1.000, providing laboratory support for the screening, diagnosis, and treatment of ICP.
[0020] In the early stages of this invention, a peptidomics approach was used to obtain the small molecule peptide QGAKIPKPEAS specifically and abnormally expressed in the blood of ICP patients, and MRM technology was used to validate it in a large sample of ICP serum. The application of the above methods and strategies accelerated and ensured the application of the serum / plasma small molecule peptide biomarker QGAKIPKPEAS diagnostic kit, and also provided a reference for the development of biomarkers for other diseases.
[0021] This invention investigates the application prospects of the serum / plasma small molecule peptide biomarker QGAKIPKPEAS in the diagnosis of ICP by controlling for factors such as age that influence disease progression. It elucidates the impact of abnormally expressed QGAKIPKPEAS on ICP progression and reveals its diagnostic value for ICP. Therefore, this invention obtains a serum / plasma small molecule peptide biomarker specific to ICP pathogenesis. Through the development and application of the serum / plasma small molecule peptide biomarker QGAKIPKPEAS and diagnostic kits, the diagnosis of ICP can be made more convenient and easier, laying the foundation for clinicians to quickly and accurately diagnose ICP and implement treatment measures. It also helps in the discovery of novel small molecule drug targets with potential therapeutic value. Attached Figure Description
[0022] Figure 1 Diagnostic value of serum QGAKIPKPEAS for ICP Detailed Implementation
[0023] Example 1: Collection of Study Samples and Data Processing
[0024] The inventors collected a large number of peripheral blood samples from ICP patients and healthy pregnant controls (samples used in the study were collected at the same time, and the sampling, aliquoting, and storage conditions were uniform). Through the collation of sample data, the inventors selected 60 samples that met the following criteria as experimental samples for peptidomics detection based on TMT labeling and LC-MS / MS mass spectrometry and for validation based on MRM technology:
[0025] (1) Case group included: The diagnostic criteria for ICP were based on the ICP Patient Diagnosis and Treatment Guidelines (First Edition), specifically as follows: 1) Skin itching during mid-to-late pregnancy, or accompanied by varying degrees of jaundice; 2) Laboratory tests: elevated serum total bile acid (TBA) (>40 μmol / L), or accompanied by mild to moderate elevation of transaminases (ALT and AST), possibly with elevated bilirubin; 3) Pregnancy was the sole cause of skin itching and biochemical abnormalities; 4) The patient was in good general condition, without significant vomiting, poor appetite, weakness, or other symptoms; 5) The above symptoms, signs, and serum biochemical indicators rapidly returned to normal after delivery. Thirty ICP patients with complete clinical data were included.
[0026] (2) Included normal control group: 30 normal pregnant women with complete clinical data who had no pregnancy complications and comorbidities, whose indications for cesarean section were breech presentation, pelvic abnormalities and social factors.
[0027] (3) Exclusion criteria for two groups: 1) other hepatobiliary diseases; 2) other pregnancy complications such as gestational hypertension or blood, urine or biochemical abnormalities that cannot be explained by ICP; 3) systemic diseases such as diabetes, hypertension, mental and neurological diseases; 4) hereditary or immune diseases; 5) history of blood transfusion, transplantation or immunotherapy; 6) history of oral contraceptives.
[0028] This study used 60 eligible samples and systematically collected demographic and clinical data from these samples.
[0029] Example 2: Differential Peptide Profiling Analysis of ICP Patients
[0030] Five ICP cases and five healthy controls meeting the above criteria were analyzed using peptidomics to obtain relevant results. The specific experimental methods are as follows:
[0031] 1.1 Instruments
[0032] Nanoscale liquid chromatography EASY-nLC 1200 System (ThermoFisher Scientific); High-resolution mass spectrometer QExactive HF-X (ThermoFisher Scientific); High-speed low-temperature centrifuge (Beckman Instruments, Germany); General centrifuge (Eppendorf, Germany); Constant temperature water bath (Shanghai Senxin Experimental Instrument Co., Ltd.); NanoDrop@ND-1000 spectrophotometer (NanoDrop, USA); Electronic analytical balance (Changshu Shuangjie Test Instrument Factory); Pure water & ultrapure water system (Millpore, USA); Ultrafiltration centrifuge tubes (10 kDa) (Millpore, USA); C18 purification column (Waters); Analytical column 0.075 × 150 mm (ThermoFisher Scientific); Concentrator (Labconco CentriVap).
[0033] 1.2 Reagents
[0034] Urea (GibcoBRL); Acetic acid (Sigma); Double-distilled water (prepared using a MilliQ pure water system); Formic acid (Sigma); Acetonitrile (Fisher Scientific); Methanol (Fisher Scientific); TMT Mass Tagging Kits and Reagents-10plex (ThermoFisher); Dithiolitol (DTT) (Promega); Iodoacetamide (IAM) (Promega); TEAB (Sigma); Trifluoroacetic acid (TFA) (Sigma); Protease inhibitor (Roche, Switzerland).
[0035] 1.3 Extraction of blood polypeptides
[0036] Take the same volume of each sample, add a certain volume of lysis buffer containing protease inhibitors, vortex, centrifuge at 20000g for 15 minutes at 4℃, and collect the supernatant; reduce to a final concentration of 5mM DTT, incubate at 56℃ for 1 hour; alkylate to a final concentration of 14mM IAM, and react in the dark for 30 minutes; use an ultrafiltration tube with a molecular weight cutoff of 10 kDa (kilodaltons), moisten with 1 ml of sterile water before use, set the temperature to 4℃, centrifuge at 14000g for 5 minutes to check the patency and integrity of the ultrafiltration tube membrane; then add protein extraction buffer, centrifuge at 14000g for 15 minutes until no more effluent is filtered out, and collect the effluent.
[0037] 1.4 Purification and Quantification of Blood Polypeptides
[0038] The peptide sample was pH adjusted to acidic; 1 ml of anhydrous acetonitrile was added to a C18 column; 1 ml of 50% acetonitrile and 0.5% acetic acid were added to activate the column; 1 ml of 0.1% TFA was added to equilibrate the column; the peptide sample was added and loaded twice; 1 ml of 0.1% TFA was added to remove impurities; 1 ml of 50% acetonitrile and 0.5% acetic acid was added to elute, and the elution was collected; the sample was lyophilized. The concentration of the peptide was detected at a wavelength of 280 nm using a NanoDrop instrument (Thermo NanoDrop 2000 micro-volume UV spectrophotometer).
[0039] Labeling of 1.5 peptides
[0040] Before use, equilibrate the TMT10 tagging reagent to room temperature and dissolve the tagging reagent in anhydrous acetonitrile; add the TMT10 tagging reagent to 10 peptide samples respectively; incubate at room temperature for 1 hour; add 5 μL of 5% hydroxylamine to the sample and incubate for 15 minutes to terminate the reaction; mix the 10 samples in equal volumes and freeze-dry.
[0041] 1.6 Liquid chromatography-mass spectrometry (LC-MS / MS)
[0042] The peptides were analyzed using an online nano-level liquid chromatography system, EASY-nLC 1200 System (ThermoFisher Scientific), for separation, and then detected by a Q Exactive HF-X Mass Spectrometer (ThermoFisher Scientific) in series.
[0043] 1.6.1 Chromatographic conditions: Mobile phase: A, 100% water, 0.1% formic acid; B, 80% acetonitrile, 0.1% formic acid. Analytical column: C18, 0.075*150mm, 1.9µm. Flow rate: 300 nmol / min. Chromatographic elution gradient is shown in Table 1.
[0044] Table 1 Elution gradient at 180 min
[0045]
[0046]
[0047] 1.6.2 Mass Spectrometry Conditions: Instrument: Q Exactive HF-X Mass Spectrometer (ThermoFisherScientific); First-stage acquisition (Full scan MS) resolution 60,000, AGC target 3e6, maximum IT time 80ms; Second-stage acquisition (dd-MS2) resolution 45,000, AGC target 1e5, maximum IT time 80ms; NCE 32, dynamic exclusion 30s.
[0048] 1.7 Database retrieval and bioinformatics analysis
[0049] The qualitative search parameters are as follows:
[0050] Table 2 List of search parameters
[0051]
[0052] Fixed modification refers to the fixed modification; Carbamidomethyl (C) is the urea methylation of cysteine after reductive alkylation. Variable modification refers to the variable modification; Oxidation (M) is the oxidation of methionine; TMT10plex (K) and TMT10plex plex (N-term) are the binding of TMT reagents at different positions. peptide tol is the precision of primary mass spectrometry. MS / MS tol is the precision of secondary mass spectrometry. Database is the database used for searching.
[0053] The quantitative analysis parameters are as follows:
[0054] Table 3 List of Quantitative Parameters
[0055]
[0056] Protein ratio Type: The peptide value selection method used for quantification; "median" refers to the method of taking the median position. Minimum peptides: The minimum number of unique peptides used for quantification. Normalization method: The correction method; "median" refers to selecting the median value of all quantifiable proteins in a sample for correction. P-value: Protein confidence assessment; less than 0.05 indicates a protein confidence greater than 95%. Fold difference refers to the direct ratio of reporter group strengths; in this experiment, 1.3 fold was selected as the fold difference threshold.
[0057] 1.8 Results of Peptide Omics Analysis
[0058] For statistical significance analysis, data with at least half of the data points in each group being non-empty were selected. Metabolites with an expression fold greater than 1.3-fold (up- and down-regulation) and a P-value (ttest) less than 0.05 were screened as differentially expressed small peptides. In 5 ICP cases and 5 healthy controls, we initially screened 629 small peptides using TMT labeling and LC-MS / MS mass spectrometry, among which 66 small peptides showed significant differences, with QGAKIPKPEAS expression being significantly reduced.
[0059] Example 3: Verification of the expression of the small molecule peptide marker QGAKIPKPEAS in ICP serum / plasma using MRM mass spectrometry.
[0060] Thirty ICP patients and 30 healthy controls meeting the criteria in Example 1 were selected. The expression levels of the small molecule peptide marker QGAKIPKPEAS in serum / plasma samples from both groups were quantitatively detected using MRM mass spectrometry. Differences were analyzed and compared, and ROC curves were plotted. The specific method is as follows:
[0061] 1.1 Instruments
[0062] High-speed low-temperature centrifuge (Beckman Instruments, Germany); ordinary centrifuge (Eppendorf, Germany); -20℃ freezer (Haier); constant temperature water bath (Shanghai Senxin Experimental Instrument Co., Ltd.); NanoDrop@ND-1000 spectrophotometer (NanoDrop, USA); ice maker (SIM-F124 model) (SANYO Ltd., Japan); analytical balance (Changshu Shuangjie Test Instrument Factory); pure water & ultrapure water system (Millpore, USA); ultrafiltration centrifuge tubes (10 kDa) (Millpore, USA); C18 purification column (Waters); Sciex LC (AB Sciex); AB Sciex 5500 (Q-TRAP-MS) (ABSciex); BEH-C18 column (1.7 μm, 2.1 × 100 mm) (Waters); concentration system (Labconco CentriVap).
[0063] 1.2 Reagents
[0064] Urea (GibcoBRL); Acetic acid (Sigma); Double-distilled water was prepared in our laboratory using a MilliQ water purifier; Formic acid (Sigma); Acetonitrile (Fisher Scientific); Methanol (Fisher Scientific); Dithiothreitol (DTT) (Promega); Iodoacetamide (IAM) (Promega); Trifluoroacetic acid (TFA) (Sigma); Protease inhibitor (Roche).
[0065] 1.3 Peptide Synthesis
[0066] The target peptide was selected and synthesized, and then dissolved in 15% ACN + 85% H2O to verify the synthesis information.
[0067] 1.4 Optimization of target peptide ion pairs
[0068] The target peptide was selected from the two ion pairs with the strongest signals: the first and third ion pairs of 786-3, as shown in the table below:
[0069] Table 4 Information on target peptide ion pairs
[0070]
[0071] 1.5 Construction of Standard Curve Using External Standard Method
[0072] Analyte Name: 786-3_1
[0073] Regression Equation:y=7253.87941x+-2384.89703(r=0.99900,r 2 =0.99799)(weighting:1 / x)
[0074] Table 5. Information on the preparation of the standard curve for QGAKIPKPEAS (786-3_1)
[0075]
[0076] AnalyteName:786-3_3
[0077] Regression Equation:y=3041.34704x+34.10897(r=0.99837,r 2 =0.99675)(weighting:1 / x)
[0078] Table 6. Information on the preparation of the standard curve for QGAKIPKPEAS (786-3_3)
[0079]
[0080]
[0081] 2.6 Extraction and purification of blood polypeptides: Same as 1.3 and 1.4 in Example 2.
[0082] 2.7 Mass spectrometry Multiple Reaction Monitoring (MRM) Analysis
[0083] 2.7.1 Chromatographic conditions: See the table below.
[0084] Table 7 Chromatographic reaction conditions for MRM technology
[0085]
[0086] 2.7.2 Mass spectrometry conditions: The instrument used was an ABSciex 5500 triple quadrupole tramp mass spectrometer (Q-TRAP-MS). Specific parameters are shown in the table below.
[0087] Table 8 Mass spectrometry reaction parameters of MRM technology
[0088]
[0089] 1.8 Data Analysis
[0090] The content of the target peptide was calculated using a standard curve based on the signal intensity or peak area of each sample in mass spectrometry. Finally, statistical analysis was performed on the expression of the experimental and control groups. The inventors detected QGAKIPKPEAS in serum samples from 30 ICP patients and 30 healthy controls during pregnancy in an independent population, and plotted ROC curves. Student's t-test was used to compare differences in demographic characteristics, TBA (μmol / L) levels, and the average expression level of the small molecule peptide marker QGAKIPKPEAS in serum / plasma samples among the study groups. Statistical analysis was performed using SPSS 22.0 software. The statistical significance level was set at 0.05, and all statistical tests were two-tailed. The results are shown in Table 9.
[0091] Table 9 Comparison of serum QGAKIPKPEAS detection results between ICP patients and normal controls.
[0092]
[0093] **P<0.01
[0094] Based on the validation experiment of serum small molecule peptide markers, the inventors detected a significant difference in the expression of the small molecule peptide marker QGAKIPKPEAS in the serum of the "ICP case" group (30 cases) and the "healthy female control" group (30 cases) (P<0.01). Multivariate logistic regression analysis showed that QGAKIPKPEAS was significantly associated with the pathogenesis of ICP, and its ROC curve is shown in the figure. Figure 1 The AUC of QGAKIPKPEAS was 1.000, indicating that QGAKIPKPEAS is a small molecule peptide biomarker that is highly correlated with the pathogenesis of ICP and can be used to prepare ICP auxiliary diagnostic kits.
[0095] Example 4: Kit for ICP Disease Diagnosis
[0096] This kit includes reagents and consumables for the detection of QGAKIPKPEAS small molecule peptides, including the synthetic peptides of QGAKIPKPEAS. Other components include a chromatographic column (BEH-C18 column, 1.7 μm, 2.1 × 100 mm) for MRM technology, urea, acetic acid, double-distilled water, formic acid, acetonitrile, methanol, dithiothreitol, iodoacetamide, trifluoroacetic acid, and protease inhibitors. The value of this kit lies in its ability to detect serum QGAKIPKPEAS levels using only 400 μl of pregnant plasma, enabling diagnosis of ICP based on these levels and facilitating dynamic monitoring and observation of treatment efficacy.
[0097] The specific reagent kit consists of the following components:
[0098] Synthetic peptides of QGAKIPKPEAS
[0099] Chromatographic column (BEH-C18 column, 1.7 μm, 2.1 × 100 mm)
[0100] Urea, acetic acid, double-distilled water, formic acid, acetonitrile, methanol, dithiothreitol, iodoacetamide, trifluoroacetic acid, and protease inhibitors.
Claims
1. Use of a polypeptide as shown in SEQ ID NO. 1 as a detection target in the preparation of a serum / plasma auxiliary diagnostic kit for intrahepatic cholestasis of pregnancy.
2. Use of a reagent for quantitatively detecting a polypeptide as shown in SEQ ID NO. 1 in the preparation of a serum / plasma auxiliary diagnostic reagent for intrahepatic cholestasis of pregnancy.