CN117517677A - Biomarker for heart failure phlegm stasis syndrome and application thereof - Google Patents

Biomarker for heart failure phlegm stasis syndrome and application thereof Download PDF

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Publication number
CN117517677A
CN117517677A CN202311528620.8A CN202311528620A CN117517677A CN 117517677 A CN117517677 A CN 117517677A CN 202311528620 A CN202311528620 A CN 202311528620A CN 117517677 A CN117517677 A CN 117517677A
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heart failure
stasis syndrome
phlegm stasis
phlegm
biomarker
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吕渭辉
蓝涛华
江巍
李晓庆
许文静
张曈
刘彤
朱珲莹
曹海明
姚萍
黄桂宝
屈玉春
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Guangdong Hospital of Traditional Chinese Medicine
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Guangdong Hospital of Traditional Chinese Medicine
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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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
    • G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
    • G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
    • G01N2333/96411—Serine endopeptidases (3.4.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Abstract

The invention discloses a biomarker for heart failure phlegm stasis syndrome and application thereof, wherein the biomarker is selected from at least one of IGFBP4, B2M, DAG1, J CHAIN and KLKB 1. The heart failure phlegm stasis syndrome biomarker provided by the invention is applied to clinical diagnosis or auxiliary diagnosis of heart failure phlegm stasis syndrome patients, has higher sensitivity and specificity, can be used for accurately identifying heart failure phlegm stasis syndrome patients, and improves the accuracy of heart failure clinical diagnosis.

Description

Biomarker for heart failure phlegm stasis syndrome and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a heart failure phlegm stasis syndrome biomarker and application thereof in preparation of a product for diagnosing or assisting in diagnosing heart failure phlegm stasis syndrome.
Background
Chronic heart failure (abbreviated as "chronic heart failure") is a complex set of clinical syndromes caused by abnormal changes in heart structure and function, which are caused by various causes, and dysfunction of the ventricles, which are serious manifestations and late stages of various heart diseases. Despite standard western medicine treatment under guidance, there are still a large number of patients suffering from heart failure progression, recurrent episodes of acute heart failure, frequent hospitalization, poor quality of life, and the like. The treatment of heart failure such as CRT, ICD or left ventricular assist device is expensive, and patients who meet the implant indication need strict screening, which is not suitable for all heart failure patients. Currently, heart failure incidence, the death rate of heart failure patients during hospitalization and the death rate of heart failure patients in 5 years are still maintained at higher levels, heart failure becomes a serious public health problem seriously harming national health and bringing heavy economic burden to society, and elucidation of pathophysiological mechanisms and searching for more effective treatment strategies as soon as possible become important points of current researches.
The diagnosis and treatment of chronic heart failure by combining the symptoms and signs has the characteristic of combining the accuracy and individuation, and simultaneously enriches the means of heart failure treatment. The syndrome (simply referred to as "syndrome") is a disease classification method established according to the theory of traditional Chinese medicine, namely, the generalization of comprehensive expression of organisms under different individuals, different stages, different environments and the like in one or more disease states, and is an "objective existence". The traditional Chinese medicine considers that heart failure is caused by deficiency and excess, and the deficiency is mainly the deficiency of heart, lung, spleen and kidney; the excessive symptoms are complicated by blood stasis and phlegm-dampness. The scholars explore the distribution rule of the traditional Chinese medicine symptoms of heart failure through methods such as literature research, expert questionnaires or clinical epidemiological investigation, and the specific results of each research are different, but the specific results show inherent consistency, namely qi deficiency, phlegm dampness and blood stasis are the most common symptoms of heart failure. Investigation of diastolic heart failure patients shows that the disease position is mainly concentrated in heart, lung and kidney, the disease symptoms are mainly qi deficiency, yang deficiency, dampness and phlegm, and the disease symptoms are 64.5%, 63.1%, 43.3% and 62.4% respectively. In recent 10 years, the literature analysis of traditional Chinese medicine symptoms related to chronic heart failure shows that the traditional Chinese medicine symptoms gradually increase along with the deterioration of heart function, but qi deficiency and blood stasis are still main symptoms, and the proportion of patients with yang deficiency and water retention symptoms in the class IV heart function increases. Therefore, the differentiation of heart failure in traditional Chinese medicine has a certain rule and can be followed. The principal symptoms of excess, blood stasis and phlegm dampness, are often the main clinical manifestations of heart failure and transformation of the disease condition, and the two are often stuck and causally mutually caused, and are difficult to remove after long-term stay, and persist. If the phlegm stagnation factor in the heart failure is accurately found and targeted treatment is carried out, the accuracy of heart failure syndrome differentiation can be obviously improved, and the curative effect of clinical treatment can be improved.
The "syndrome" (or syndrome symptoms) is the center of the theory of "treatment based on syndrome differentiation" in TCM. However, for a long time, the lack of objectively quantized syndrome judging indexes and syndrome curative effect evaluating indexes has been the bottleneck which plagues the modernization and objectification of the syndrome of traditional Chinese medicine. At present, objective researches on the common disease element of heart failure phlegm stasis syndrome are still lacking, and biomarkers of heart failure phlegm stasis syndrome are not reported yet; diagnostic strategies and key technologies for heart failure and phlegm stasis syndrome combination are not established.
Disclosure of Invention
The invention aims to provide a specific biomarker for heart failure phlegm stasis syndrome. The specific biomarker provided by the invention has higher sensitivity and specificity, can be applied to diagnosis or auxiliary diagnosis of heart failure phlegm stasis syndrome, and is used for accurate identification of heart failure phlegm stasis syndrome patients.
According to one aspect of the present invention there is provided the use of a biomarker selected from at least one of insulin-like growth factor binding protein 4 (igfbp 4), β2-Microglobulin (B2M), dystrophin-associated glycoprotein 1 (Dystrophin Associated Glycoprotein 1, dag 1), jchan, kallikrein B1 (Kallikrein 1B, klkb 1) in the manufacture of a product for diagnosing or aiding in the diagnosis of heart failure phlegm stagnation syndrome.
The results of the target Proteomics (PRM) verification show that the content of IGFBP4, B2M, DAG1, JCAIN and KLKB1 in the peripheral blood serum of patients with heart failure phlegm stasis syndrome is obviously different from the content of IGFBP4 in the peripheral blood serum of healthy people and patients with heart failure non-phlegm stasis syndrome. The sensitivity and the specificity of the 5 proteins IGFBP4, B2M, DAG1, J CHAIN and KLKB1 for predicting the heart failure phlegm stasis syndrome are evaluated by adopting a ROC curve, and the results show that AUC values of the IGFBP4, the B2M, DAG1 are respectively 0.79, 0.8, 0.85 and 0.826, which indicate that the 5 proteins provided by the invention have higher sensitivity and specificity for diagnosing the heart failure phlegm stasis syndrome, can effectively distinguish heart failure phlegm stasis syndrome patients from healthy people, and can be used as specific biomarkers of heart failure phlegm stasis syndrome.
The heart failure phlegm stasis syndrome biomarker provided by the invention can provide reliable basis and practical guidance for clinical diagnosis and drug treatment of heart failure phlegm stasis syndrome, and is beneficial to realizing individuation and accurate treatment of chronic heart failure diagnosis and treatment.
In the invention, the 5 proteins IGFBP4, B2M, DAG1, J CHAIN and KLKB1 can be independently used as biomarkers for diagnosing or assisting in diagnosing whether a patient is a patient with heart failure and phlegm stasis, the kit can also be used as a biomarker for diagnosing or assisting in diagnosing whether a patient is a heart failure phlegm stasis syndrome patient or not by combining two or more of the above-mentioned materials, and can be applied to preparing a product capable of diagnosing or assisting in diagnosing heart failure phlegm stasis syndrome.
In the present invention, products for diagnosing or aiding in diagnosing heart failure phlegm stasis syndrome include, but are not limited to, reagents, kits, chips or instruments for diagnosing or aiding in diagnosing whether a subject is a patient with heart failure phlegm stasis syndrome. Heart failure with phlegm stagnation can be diagnosed by detecting the level of at least one of the 5 proteins IGFBP4, B2M, DAG1, J-chann, KLKB1 in the serum of the subject, if there is an elevated level of IGFBP4, B2M, DAG, or a reduced level of J-chann, KLKB1, at least 1 of the above indices are met.
According to another aspect of the invention, there is provided the use of a product for detecting the level of a heart failure phlegm stasis syndrome biomarker in a sample in the manufacture of a product for diagnosing or aiding in the diagnosis of heart failure phlegm stasis syndrome, wherein the heart failure phlegm stasis syndrome biomarker is selected from at least one of IGFBP4, B2M, DAG1, JCHAIN, KLKB 1.
In the present invention, the product for detecting the biomarker content of heart failure in a sample can be a product for detecting the protein content of IGFBP4, B2M, DAG1, J CHAIN, or KLKB1 in a sample by any technique known in the art that can detect the protein content in a sample, including but not limited to reagents, kits, chips, and/or instruments suitable for detecting the protein content of the relevant protein by at least one of the following methods: western blotting, enzyme-linked immunosorbent technique, mass spectrometry, protein chip technique, parallel reaction monitoring technique or proteomics technique.
Specifically, the product for detecting the content of the relevant protein in the sample can be a mass spectrometer, an ELISA protein detection reagent or kit, a Western blotting reagent or kit and the like.
In some embodiments, the sample is peripheral blood serum.
In some embodiments, the product for detecting the level of heart failure sputum stasis syndrome biomarkers in peripheral blood serum may be a reagent, kit, chip and/or instrument suitable for detecting the level of heart failure sputum stasis syndrome biomarkers in peripheral blood serum by enzyme-linked immunosorbent technique.
In some embodiments, the product for detecting the content of the heart failure phlegm stasis syndrome biomarker in the peripheral blood serum can be a kit suitable for detecting the content of the heart failure phlegm stasis syndrome biomarker in the peripheral blood serum by an enzyme-linked immunosorbent technique.
In some embodiments, the kit may be an ELISA protein detection kit. Besides the antibodies to the corresponding proteins, other components such as buffer solution, washing solution, dilution solution, blocking solution, stop solution, color developing solution, etc. may be included in the kit, and those skilled in the art may select appropriate components and amounts according to actual needs, which are not particularly limited in the present invention.
The invention provides a plurality of specific biomarkers for identifying patients with heart failure phlegm stasis syndrome, and the biomarkers are applied to clinical diagnosis or auxiliary diagnosis of the patients with heart failure phlegm stasis syndrome, have higher sensitivity and specificity, can provide reliable basis and practical guidance for clinical diagnosis and drug treatment of heart failure phlegm stasis syndrome, improve the accuracy of clinical diagnosis of heart failure, are beneficial to realizing individuation and accurate treatment of chronic heart failure, and further improve the clinical curative effect of traditional Chinese medicine for treating heart failure.
Drawings
FIG. 1 is a baseline data for each group of patients;
FIG. 2 is a PCA plot of the proteins of the HF-TY group, the HF group and the healthy control group;
FIG. 3 is a volcanic plot of the differential protein of the HF-TY group versus the healthy control group;
FIG. 4 is a volcanic plot of differential protein between the HF group and the healthy control group;
FIG. 5 is a volcanic plot of the differential proteins of the HF-TY group and the HF group;
FIG. 6 is a heat map of the first 50 differential proteins of the HF-TY group, the HF group and the healthy control group;
FIG. 7 is a view of the results of a Wien analysis;
FIG. 8 shows the top 10 GO-enriched entries related to biological processes, cellular components and molecular functions for the HF-TY group differentially expressed proteins from healthy control group;
FIG. 9 is a KEGG pathway analysis of differentially expressed proteins between the HF-TY group and the healthy control group;
FIG. 10 is a bubble diagram of the first 20 KEGG pathways of the HF-TY group and healthy control group;
FIGS. 11 to 15 are box plots of 10 validated protein expression levels for the HF-TY group, the HF group and the healthy control group;
FIG. 16 is a box-shaped diagram of JCHAIN, IGHM, KLKB, IGFBP4, B2M and DAG1 in DIA and PRM assays;
FIGS. 17-21 show ROC curves of HF-TY group versus healthy controls, B2M, DAG, IGFBP4, JCAIN, KLKB 1.
Detailed Description
The invention will be described in further detail with reference to specific embodiments and drawings. The examples are for illustration only and are not intended to limit the invention in any way. Unless otherwise indicated, reagents used in the examples were prepared as commercially available products and experimental procedures under conditions not specified in the examples were prepared as conventional procedures and conditions well known in the art or as suggested by the manufacturers of the kits and instruments.
Example 1
1. Case sources
The cases are selected from community health crowd, and outpatient service or inpatient in Guangdong province, including health crowd of physical examination and people conforming to related disease diagnosis.
2. Disease diagnosis criteria
(1) Diagnostic criteria for sputum syndrome in TCM:
the diagnosis standard of the phlegm syndrome refers to the diagnosis standard of the traditional Chinese medicine which is issued by the specialized committee of the phlegm syndrome of the society of Chinese medicine in the 2016 world.
Main symptoms are as follows: greasy coating (3 points), heaviness of the head and body (3 points);
secondary symptoms: slippery pulse (2 minutes), expectoration (2 minutes), snoring (1 minute), chest and abdomen fullness (1 minute), dizziness (1 minute);
objectification index: BMI >28 (3 min), TC >5.72mmol/L or TG >1.70mmol/L or LDL >3.64mmol/L (1 min).
The diagnosis condition is that 1 main symptom and 1 secondary symptom are only present or the integral is more than or equal to 4 minutes, and the phlegm syndrome can be diagnosed.
(2) Blood stasis syndrome diagnostic criteria:
the blood stasis diagnosis standard refers to the practical blood stasis diagnosis standard issued by the specialized committee for activating blood and dissolving stasis of the Chinese and Western medicine combination society of 2016.
The main standard is as follows: (1) dark purple tongue or ecchymosis and ecchymosis; (2) Facial, lips, gums, periocular and finger (toe) end bluish violet or dark; (3) Varicose veins or abnormal telangiectasias at different sites; (4) Menstrual blood (blood stasis, blood accumulation in organs, tissues, subcutaneous or serosal cavities after bleeding); (5) intermittent claudication; (6) abdominal pain resistance; (7) amenorrhea or dark menstrual blocks; (9) Imaging shows objective evidence of vascular occlusion or moderately severe stenosis (. Gtoreq.50%), thrombosis, infarction or embolism, or visceral ischemia.
Secondary standard: (1) Fixed pain, or stinging, colic, or pain especially during the night; (2) numbness of the extremities or hemiplegia; (3) dysmenorrhea; (4) Skin manicuring (rough, hypertrophic, scaling); (5) mania or amnesia; (6) pulse astringency or substitution, or no pulse; (7) Enlargement of viscera, neoplasms, inflammatory or non-inflammatory masses, and tissue hyperplasia; (8) Imaging and other examinations show vascular stenosis (< 50%); (9) Physical and chemical detection abnormalities of rheological property, coagulation, fibrinolysis, microcirculation and the like of blood indicate blood circulation stasis; (10) trauma, surgery or induced abortion in the vicinity of 1 month.
1 or 2 secondary standards are met, and the blood stasis syndrome can be diagnosed.
Each of the main standard is divided into 2 points, and each of the secondary standard is divided into 1 point, so that the blood stasis syndrome quantitative diagnosis standard can be used.
(3) Heart failure diagnostic criteria:
the heart failure diagnosis aspect refers to the guidelines for diagnosis and treatment of heart failure of China, the society of cardiovascular diseases, the China society of medical science, the 2013AHA/ACC, the guidelines for management of heart failure, the 2016ESC, the guidelines for diagnosis and treatment of acute and chronic heart failure, and the diagnosis is mainly comprehensively judged by comprehensive clinical manifestations (symptoms and signs) and auxiliary examination (heart color Doppler ultrasound, BNP/NT-proBNP).
3. Inclusion criteria
(1) The age is 18-80 years.
(2) Meets the relevant diagnostic criteria.
(3) And signing an informed consent form.
4. Exclusion criteria
(1) Meets the relevant diagnostic criteria, but cannot provide specific data, or the past data does not meet the relevant diagnostic criteria.
(2) Other specific diseases such as conscious disturbance (somnolence, comatose, coma, confusion, delirium, unconsciousness), mental diseases (Alzheimer's disease, schizophrenia, major depression, mania) are not well understood by the study item and others who cannot be matched with syndrome differentiation.
(3) Complications such as level IV cardiac dysfunction, severe arrhythmia (rapid atrial fibrillation, ventricular rate > 160 beats/minute, atrial flutter) affect the differentiation of the disease and the collection of data.
(4) Liver and kidney dysfunction (ALT, AST 2 times higher than the upper limit, or serum creatinine > 265. Mu. Mol/L).
(5) Pregnant or lactating women.
(6) Familial hyperlipidemia.
(7) Secondary hyperlipidemia caused by specific drugs (e.g., diuretics, beta-blockers, glucocorticoids, estrogens, etc.) or other diseases.
(8) Patients suffering from hyperlipidemia are required to take the medicine recently or for a long period of time.
(9) Patients with poor compliance and unable to review on time are expected.
5. DIA proteomics primary screening
5.1 sample preparation
Grouping standard:
healthy control group (health controls/CON): the standard of modern medicine of healthy people is met, the integral of phlegm syndrome is less than or equal to 2 points, and the diagnosis standard of blood stasis syndrome is not met;
heart failure non-phlegm stasis syndrome group (HF): the heart failure diagnosis is met, the integral of the phlegm syndrome is less than or equal to 2 minutes, and the blood stasis syndrome diagnosis standard is not met;
heart failure phlegm stasis syndrome group (HF-TY): the heart failure and blood stasis syndrome diagnosis is met, and the integral of the phlegm syndrome is more than or equal to 6 minutes.
The number of initial screening serum samples was assigned to 5 groups, and baseline data for each group of patients is shown in FIG. 1.
5mL venous blood is taken in each case, kept stand for 2 hours at room temperature, centrifuged for 10 minutes at 4 ℃ and 1500 Xg, and the supernatant is taken and split-packed and stored in a refrigerator at-80 ℃ for standby.
5.2DIA proteomic detection
(1) Protein extraction and assay
An albumin/IgG high abundance protein removal kit (manufacturer Millipore, cat No. 122642) was used. The method comprises the following specific steps:
40. Mu.L of serum was taken for each sample and diluted ten times with binding buffer. The column was removed from the lid and the storage buffer was blotted with paper. Removing the sharp mouth at the bottom of the column, and placing into a collecting pipe with proper size. Binding buffer was added and allowed to flow through the cartridge by gravity. The column was placed in a new, appropriately sized collection tube. Diluted serum samples were added and allowed to flow through the cartridge by gravity. The cartridge was washed with 600. Mu.L of binding buffer. And (3) washing the column again with 600 mu L of binding buffer solution, and collecting the elution components in the last three steps, namely, obtaining the sample after albumin/IgG is removed, and performing vacuum freeze drying for later use.
The lyophilized samples were reconstituted by lysis with 300. Mu.L SDS. The solution was centrifuged at 12000 Xg for 10min at room temperature, and the supernatant was collected by repeating the centrifugation once. The supernatant is the total protein solution of the sample, and is stored at-80 ℃ for standby after protein concentration measurement and split charging.
(2) Proteolysis and peptide fragment desalination
Protein concentration was measured using BCA method, 50 μg of protein per sample was added to DTT to give a final concentration of 5mM, mixed well, incubated at 55 ℃ for 30min, and then cooled on ice to room temperature. Then, the corresponding volume of iodoacetamide was added to give a final concentration of 10mM, mixed well and left at room temperature for 15min in the dark. Adding 6 times of acetone, standing at-20deg.C for four timesAbove, the pellet was collected by centrifugation at 8000 Xg at 4℃for 10 minutes. After 2-3min of volatilizing the acetone, 100. Mu.L of 50mM NH was added 4 HCO 3 The pellet was reconstituted, 1mg/mL pancreatin Trypsin-TPCK was added at 1/50 of the sample mass, and digested overnight at 37 ℃. Freeze-drying the sample after enzymolysis and storing at-80 ℃.
And desalting the peptide fragments after enzymolysis by adopting an SOLASPE 96-well plate. The column was activated with 200 μl methanol and repeated 2 more times. The column was then activated with 200. Mu.L of purified water (0.1% formic acid) and repeated 2 more times. A sample was added in a volume of 50-500. Mu.L, vacuum was adjusted and the drop velocity was maintained at 1mL/min (about 1 drop/sec). The sample is repeated once. 200 μl of 0.1% formic acid-water wash was used and repeated 2 more times. Finally, 150. Mu.L of 50% acetonitrile-water (containing 0.1% formic acid) was used to elute the peptide fragment, and the procedure was repeated 2 times for 3 times to obtain 450. Mu.L of eluent, followed by vacuum evaporation.
(3) DDA library construction
Before mass spectrum sample introduction, each sample is mixed according to the volume ratio of iRT:to-be-detected sample=1:10, and the mixture is used as an internal standard. All samples after enzymatic hydrolysis were mixed with equal amounts of peptide fragments and component separation was performed in the mobile phase at ph=10 using Agilent 1100HPLC system.
Chromatographic column: agilent Zorbax Extend-C18 narrow diameter column, 2.1X105 mm,5 μm.
Detection wavelength: ultraviolet 210nm and 280nm.
Mobile phase a phase: ACN-H 2 O (2:98, v/v), mobile phase B phase: ACN-H 2 O (90:10, v/v) (both mobile phases were pH adjusted to 10 with ammonia) at a flow rate of 250. Mu.L/min.
Gradient elution conditions: 0-10min,2% B;10-10.01min,2-5% B;10.01-37min,5-20% B;37-48min,20-40% B;48-48.01min,40-90% B;48.01-58min,90% B;58-58.01min,90-2% B;58.01-63min,2% B. Beginning at 10 minutes, eluent is collected into centrifuge tubes 1-10 at intervals of one minute in sequence, and fractions are circularly collected according to the sequence of 1-10. 10 components are collected in total, vacuum freeze-dried and pumped, and the samples are stored in a frozen state for mass spectrometry.
(4) DIA detection
Each component was separated using a nanoElute liquid phase (Bruker), mobile phase a was 0.1% aqueous FA, and mobile phase B was ACN of 0.1% FA. Gradient elution conditions: 0-45min,2-22% B;45-50min,22-37% B;50-55min,37-80% B;55-60min,80%.
Peptide fragments were separated by ultra-high performance liquid phase system and then injected into a timsTOF Pro mass spectrometer (Bruker) for analysis under the following conditions: the capillary voltage is 1.4KV, the temperature of the drying gas is 180 ℃, the flow rate of the drying gas is 3.0L/min, the mass spectrum scanning range is 100-1700m/z, the ion mobility range is 0.85-1.3Vs/cm < 2 >, and the collision energy range is 20-59eV.
(5) Qualitative and quantitative and functional analysis of proteins
DIA raw data was processed using Spectronaut Pulsar software with fixed modifications of Carbamidomethyl (C), variable modifications of Oxidation (M) and actyl (N-term), and a maximum leaky cut site of 2.
The differential protein needs to meet P <0.05 and FC >1.2 or FC <1/1.2, be significantly up-regulated when P <0.05 and FC >1.2, and significantly down-regulated when P <0.05 and FC < 1/1.2.
The biological processes (Biological Process, BP), cellular components (Celluar Component, CC) and molecular functions (molecular function, MF) of the differentially expressed proteins were analyzed by their biological functions and classifications using the Gene Ontology (GO) database. The major pathways involved in differential proteins were analyzed using a KEGG (Kyoto encyclopedia of genes and genomes, KEGG) database. The differential protein interaction (protein protein interaction, PPl) was analyzed based on string databases and differential protein interaction networks were constructed.
5.3DIA proteomics Primary screening results
And selecting community health crowd and outpatient or inpatient in Guangdong province, collecting peripheral venous blood according to standard operation rules, and collecting serum samples, wherein 5 samples of the community health crowd, 5 samples of heart failure non-phlegm stasis patients and 5 samples of heart failure phlegm stasis patients are collected. And (3) performing DDA library construction and DIA mass spectrometry on serum samples by using a proteomics DIA technology, performing significant difference analysis on data with at least half of the data in each group being non-null, screening proteins with expression difference multiple of more than 1.2 times (up-down regulation) and P value (T test) of less than 0.05 as differential expression proteins, and determining candidate proteins with heart failure phlegm stagnation syndrome by combining bioinformatics GO and KEGG enrichment analysis.
The results are shown in FIGS. 2 to 10.
The results show that:
(1) PCA principal component analysis results performed on the heart failure phlegm stasis syndrome group (HF-TY), the heart failure non-phlegm stasis syndrome group (HF) and the healthy control group (con) show that there is a clear distinction between the 3 groups (FIG. 2). Volcanic charts (fig. 3 and 4) show that with FC >1.2 or <0.83 and p <0.05 as screening criteria, there were significantly up-regulated and significantly down-regulated in the HF-TY and HF groups, respectively, as compared to healthy control groups, with 71 and 56 differential proteins. As shown in FIG. 5, there were 52 differential proteins significantly up-regulated and 16 differential proteins significantly down-regulated in the HF-TY group compared to the HF group. Furthermore, as shown in FIG. 6, the differential proteins between the HF-TY group, the HF group and the con group had clear clusters, and the 2 heart failure groups clustered closer. The Venn diagram (FIG. 7) shows that there are 28 common differential proteins in the HF-TY group and 28 unique differential proteins in the HF-TY group compared to the con group.
(2) GO and KEGG analysis is performed on the differential proteins compared with the con group by the HF-TY group, and as shown in fig. 8-10, GO functions mainly comprise extracellular body, collagen-containing extracellular matrix, neutrophil degranulation, cellular protein metabolism, serine-type endopeptidase activity, antibacterial body fluid reaction, platelet degranulation and focal adhesion; the KEGG pathway is primarily enriched in complement and coagulation cascades, neutrophil extracellular trap formation, extracellular matrix receptor interactions, IL-17 signaling pathway, platelet activation, staphylococcus aureus infection, fluid shear stress and atherosclerosis, pentose phosphate pathway, salmonella infection, iron death.
6. Targeted quantitative Proteomics (PRM) validation
And collecting samples of healthy people, heart failure patients with non-phlegm stasis syndrome and heart failure phlegm stasis syndrome, wherein 20 cases are grouped according to the same grouping standard and sample collecting method. The baseline data for each group of patients is shown in figure 1.
Extracting protein from serum sample, loading desalted peptide fragment on RSLC,75μm×15cm，nanoViper，C18，3μm，the column (Acclaim, pepMap) had a flow rate of 300nl/min. Gradient of 0-82min for 90min, 5-44% B;82-84min,44-90% B;84-90min,90-100% B (buffer A: HPLC H) 2 O,0.1% formic acid; buffer B80% ACN,0.1% formic acid).
The polypeptides were transferred to the gas phase and mass chromatographed using a Q exact HF mass spectrometer (Thermo Fisher Scientific, germany). In MS1, precursor scans were obtained at 70000 resolution. The MS/MS was performed at 15000 resolution (1.2 m/z isolation window width) with AGC set to 1e5. The precursor was broken down in HCD mode with an NCE energy of 28 and a maximum injection time of 100ms. Spectral analysis was manually verified using Skyline.
The results are shown in FIGS. 11 to 16.
The results show that:
taking FC >1.2 or FC <0.67 and P <0.05 as screening criteria, PRM verifies 10 differential proteins altogether, wherein 5 differential proteins (IGFBP 4, B2M, DAG1, JCAIN, KLKB 1) have significant differences in heart failure phlegm stasis syndrome group patients and healthy people and heart failure non-phlegm stasis syndrome group patients, but have no significant differences in heart failure non-phlegm stasis syndrome group and healthy people, and the change trend is consistent with DIA results.
7. ROC Curve analysis
The sensitivity and specificity of 5 candidate proteins (IGFBP 4, B2M, DAG1, JCHAIN, KLKB 1) to predict heart failure phlegm stasis syndrome were assessed using ROC curves with the samples in the "6, targeted quantitative Proteomics (PRM) validation" described above as analytical samples.
The results are shown in FIGS. 17-21, where ROC curves show that 5 candidate proteins clearly distinguish heart failure patients with phlegm stagnation from healthy individuals. AUC values for JCHAIN, KLKB1, IGFBP4, B2M, DAG1 were 0.79, 0.8, 0.85, 0.826, respectively. The results show that IGFBP4, B2M, DAG1, JCAIN and KLKB1 have higher sensitivity and specificity, and can be used as specific biomarkers for patients suffering from heart failure phlegm stasis.
What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.

Claims (7)

1. The application of heart failure phlegm stasis syndrome biomarker in preparing a product for diagnosing or assisting in diagnosing heart failure phlegm stasis syndrome, which is characterized in that the heart failure phlegm stasis syndrome biomarker is selected from at least one of IGFBP4, B2M, DAG1, J CHAIN and KLKB 1.
2. Use of a product for detecting the content of a heart failure phlegm stasis syndrome biomarker in a sample in the preparation of a product for diagnosing or aiding in the diagnosis of heart failure phlegm stasis syndrome, characterized in that the heart failure phlegm stasis syndrome biomarker is selected from at least one of IGFBP4, B2M, DAG1, J CHAIN, KLKB 1.
3. The use according to claim 2, wherein the sample is peripheral blood serum.
4. The use according to claim 3, wherein the product for detecting the level of heart failure phlegm stasis syndrome biomarkers in the peripheral blood serum is a reagent, kit, chip and/or instrument adapted for detecting the level of heart failure phlegm stasis syndrome biomarkers in the peripheral blood serum by at least one of the following methods: western blotting, enzyme-linked immunosorbent technique, mass spectrometry, protein chip technique, parallel reaction monitoring technique or proteomics technique.
5. The use according to claim 4, wherein the product for detecting the content of the heart failure phlegm stasis syndrome biomarker in the peripheral blood serum is a reagent, a kit, a chip and/or an instrument which are suitable for detecting the content of the heart failure phlegm stasis syndrome biomarker in the peripheral blood serum by an enzyme-linked immunosorbent technique.
6. The use according to claim 5, wherein the product for detecting the content of the heart failure phlegm stasis syndrome biomarker in the peripheral blood serum is a kit suitable for detecting the content of the heart failure phlegm stasis syndrome biomarker in the peripheral blood serum by an enzyme-linked immunosorbent technique.
7. The use according to claim 6, wherein the kit is an ELISA protein detection kit.
CN202311528620.8A 2023-11-15 2023-11-15 Biomarker for heart failure phlegm stasis syndrome and application thereof Pending CN117517677A (en)

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