CN107102152B - The protein marker of urine myocardial infarction and its purposes in diagnosis and prognosis - Google Patents
The protein marker of urine myocardial infarction and its purposes in diagnosis and prognosis Download PDFInfo
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- CN107102152B CN107102152B CN201710346736.8A CN201710346736A CN107102152B CN 107102152 B CN107102152 B CN 107102152B CN 201710346736 A CN201710346736 A CN 201710346736A CN 107102152 B CN107102152 B CN 107102152B
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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
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
Abstract
The present invention relates to the protein marker of urine myocardial infarction and its purposes in diagnosis and prognosis.Specifically, the present invention relates to the indentifying substance of albumen selected from the following preparation for diagnose and/or the reagent of the myocardial infarction of prognosis subject in purposes: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, Ctsz, and combinations thereof.This diagnosis of five kinds of protein for myocardial infarction, the state of an illness and treatment detection etc. have preferable potential applicability in clinical practice.
Description
Technical field
The present invention relates to field of biotechnology, relate more specifically to the protein marker of urine myocardial infarction and its are examining
Purposes in disconnected and prognosis.
Background technique
In most industrialized countries, cardiovascular disease incidence rate and the death rate are all very high, and consume 17% payment for medical care
With.Myocardial infarction is a kind of most important cardiovascular disease, leads to a series of complication, including cardiogenic shock, ventricular fiber
Change, heart failure and recurrent ischemia[1].It is counted according to heart of Europe disease association, the death rate of myocardial infarction is about 6% to 14%[2]。
In clinical diagnosis, sensitive biomarker, electrocardiogram and image technology are widely used in myocardial infarction diagnosis.
However, electrocardiogram sensitivity is low[2]；In the biomarker of myocardial infarction, CK-MB specificity is low (0.591-0.842)[2,3]。
Highly sensitive troponin T (troponin T) is same specific low (0.54-0.85), and the low (0.61- of positive prediction rate
0.87)[4].Also, it is existing that other diseases, such as direct myocardial damage or other non-cardiac diseases equally have troponin T to rise
As[5].In addition, the lasting raising in two weeks after myocardial infarction generation of blood plasma troponin T, cannot detect lysis in time and control
Therapeutic effect[6].Another diagnostic method, Imaging Method (such as coronary angiography technology) have damage to patient health[7]。
Therefore, a kind of Non-Invasive sample acquisition is urgently developed at present, there is highly sensitive and specificity, and can be used for the heart
The clinical testing procedure of flesh infarct early diagnosis and therapy effect detection.Compared to other clinical materials, such as cardiovascular organization, cell
System, blood, urine has the advantages that sample is facilitated, Non-Invasive obtains, so being a kind of good biomarker sample
This source.In recent years, urine is not only applicable in the detection and analysis of malignant tumour marker, is also started in cardiovascular and cerebrovascular disease research
It is used[8,9]。
It is different from blood sample, containing high-abundance proteins such as less albumin and immunoglobulin in urine, can subtract
High-abundance proteins inhibit the phenomenon that in few disease markers discovery procedure.In addition, urine specimen not only includes the ingredient in blood plasma,
Also containing there are many systemic metabolism object of differential responses access, contain relatively full biological information.Based on the above advantages, using urine
Proteomics strategy opens a new window of discovery cardiovascular disease related protein.
More reaction monitorings (Multiple Reaction Monitor, MRM) are a kind of targets based on second order ms signal
Mass spectrum quantitative analysis tech can carry out absolute and relative quantitative determination simultaneously, it does not need special antibody, to overcome
Limitation based on immunization method to antibody specificity and titre.More reaction detection technologies can simultaneously determine multiple proteins
Property and quantitative analysis.
In the present invention, we collect 7 myocardial infarction patients (morbidity 12 hours in, and treatment after seven days) urine and
The urine of 7 Normal groups marks the technology of quantitative technique and two-dimensional liquid chromatography-mass spectrometry to be divided using iTRAQ
Analysis.GO and IPA is carried out to the urine protein group differential protein identified first and carries out functional analysis, and selects 12 potential lifes
Substance markers object is developed based on target mass spectrum detection simultaneously to Antithrombin Ⅲ (Antithrombin-III) and benefit
The method that 12 kinds of protein such as body C3 (Complement C3) are analyzed the, to (morbidity 12 hours of 15 myocardial infarction patients
It is interior, and seven days after treatment) urine and the urines of 15 Normal groups analyzed, and utilize the statistical of mass spectrometric data
Analysis is as a result, discovery urine Antithrombin Ⅲ, Complement C_3,1 (Alpha-1-acid glycoprotein of α -1- acidoglycoprotein
1), the target mass spectrometric data of serum transferrin (Serotransferrin) and Ctsz (Cathepsin Z) is to 15
Example myocardial infarction and 15 Normal groups have good discrimination, and this five kinds of albumen can be after myocardial infarction treatment
Restore normal.This five kinds of potential source biomolecule markers can be applied to early diagnosis and the progression of disease, therapeutic effect of myocardial infarction
Detection, have good potential applicability in clinical practice.
Summary of the invention
In view of the demand of this field, according to some embodiments of the disclosure, the indentifying substance for providing albumen exists
Preparation for diagnose and/or the reagent of prognosis myocardial infarction in purposes, wherein the albumen be selected from following any one or its
Combination: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, Ctsz.
In specific embodiments, described to diagnose the early diagnosis for referring to myocardial infarction.The early diagnosis refers to: hair
Myocardial infarction diagnosis in disease 12 hours.
In specific embodiments, compared to normal healthy controls, the expression of albumen selected from the following improves instruction institute
Subject is stated with myocardial infarction: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, tissue egg
White enzyme Z, and combinations thereof.Normal healthy controls refer to the individual for not suffering from myocardial infarction.
In specific embodiments, the diagnosis refers to the myocardial infarction severity of monitoring subject.Specifically, right
Subject is sampled to compare with the protein level in normal healthy controls, according to the variation feelings of protein marker in Urine in Patients
Condition judges the severity of patient's myocardial infarction.Wherein, compared to normal healthy controls, the expression of albumen selected from the following is mentioned
Height indicates that the myocardial infarction of the subject deteriorates (progress): Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum
Transferrins, Ctsz, and combinations thereof.In specific embodiments, normal healthy controls, which refer to, does not suffer from myocardial infarction
Individual.
In specific embodiments, the prognosis refers to the curative effect evaluation of the myocardial infarction of subject.Specifically, it compares
Before treatment, the expression of albumen selected from the following reduces the validity for indicating the myocardial infarction treatment of the subject:
Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, Ctsz, and combinations thereof.
Indentifying substance suitable for the disclosure is Mass Spectrometric Identification reagent, antibody or its antigen-binding fragment.Specific real
It applies in scheme, antibody is monoclonal antibody.The disclosure to the source of species of monoclonal antibody there is no limit, it is any can combine it is upper
The antibody for stating albumen can be used.In specific embodiments, antigen-binding fragment includes but is not limited to: Fab, Fab',
(Fab') 2, Fv, ScFv, bispecific antibody, three specific antibodies, four specific antibodies, double-scFv.It is any to remain antigen binding
Active antibody fragment is suitable for the disclosure.
In specific embodiments, the Mass Spectrometric Identification reagent is used in more reaction monitorings.More reaction monitorings are
A kind of target mass spectrum quantitative analysis tech based on second order ms signal, can carry out absolute and relative quantitative determination simultaneously, it
Special antibody is not needed, to overcome the limitation based on immunization method to antibody specificity and titre.More reaction detection skills
Art can carry out qualitative and quantitative analysis to multiple proteins simultaneously.
In specific embodiments, the Mass Spectrometric Identification reagent is selected from: SEQ ID NO:1 and/or SEQ ID NO:2
Shown in the indentifying substance of labelled peptide of Antithrombin Ⅲ, Complement C_3 shown in SEQ ID NO:3 labelled peptide identification examination
Indentifying substance, the SEQ ID of the labelled peptide of α -1- acidoglycoprotein 1 shown in agent, SEQ ID NO:4 and/or SEQ ID NO:5
The labelled peptide of Ctsz shown in the indentifying substance of the labelled peptide of serum transferrin shown in NO:6, SEQ ID NO:7
Indentifying substance, and combinations thereof.
Labelled peptide is the peptide fragment for referring to represent some albumen, it is characterised in that exist and specificity there is only Mr. Yu's eggs
In white matter amino acid sequence.
In specific embodiments, expression is selected from protein level.
In specific embodiments, expression is measured in the urine specimen of subject.
In specific embodiments, the subject is people.
Another aspect provides a kind of for diagnosing and/or the kit or chip of prognosis myocardial infarction,
Indentifying substance comprising albumen selected from the following: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum turn iron egg
White, Ctsz, and combinations thereof.
In specific embodiments, the indentifying substance is Mass Spectrometric Identification reagent, antibody or its antigen-binding fragment, excellent
Selecting the antibody is monoclonal antibody.
In specific embodiments, the diagnosis and/or prognosis are selected from: the early diagnosis of myocardial infarction, myocardial infarction
Curative effect assessment, monitor subject myocardial infarction severity, and combinations thereof.
In specific embodiments, the Mass Spectrometric Identification reagent is used in more reaction monitorings.Specifically implementing
In scheme, the Mass Spectrometric Identification reagent is selected from: the mark of Antithrombin Ⅲ shown in SEQ ID NO:1 and/or SEQ ID NO:2
Sign indentifying substance, SEQ the ID NO:4 and/or SEQ of the labelled peptide of Complement C_3 shown in the indentifying substance of peptide, SEQ ID NO:3
Serum transferrin shown in the indentifying substance of the labelled peptide of α -1- acidoglycoprotein 1, SEQ ID NO:6 shown in ID NO:5
The indentifying substance of labelled peptide, Ctsz shown in SEQ ID NO:7 labelled peptide indentifying substance, and combinations thereof.
In specific embodiments, the kit or chip also include the indentifying substance of albumen selected from the following: white
Albumen, Annexin A1, neutrophil leucocyte alexin 1, interior acrasin 1, slightly rich proline protein 3, Delta albumen homology egg
White 1, and combinations thereof.
Specifically, kit or chip also include indentifying substance selected from the following: SEQ ID NO:8 and/or SEQ ID
The mirror of the labelled peptide of Annexin A1 shown in the indentifying substance of the labelled peptide of albumin shown in NO:9, SEQ ID NO:10
Determine shown in the indentifying substance of the labelled peptide of neutrophil leucocyte alexin 1 shown in reagent, SEQ ID NO:11, SEQ ID NO:12
The indentifying substance of labelled peptide of interior acrasin 1, slightly rich proline shown in SEQ ID NO:13 and/or SEQ ID NO:15
The identification examination of the labelled peptide of Delta albumen homology albumen 1 shown in the indentifying substance of the labelled peptide of albumen 3, SEQ ID NO:14
Agent, and combinations thereof.
Another aspect provides one kind for diagnosis and/or prognosis subject's myocardial infarction method, including
Step:
1) urine specimen of subject is obtained,
2) Urine proteins optionally, are separated from urine specimen,
3) determine the expression of albumen selected from the following in subject's urine specimen: Antithrombin Ⅲ, Complement C_3,
α -1- acidoglycoprotein 1, serum transferrin, Ctsz, and combinations thereof.
In specific embodiments, expression is determined using mass spectrometry method, ELISA method or Western method.
It, can be with after the step of obtaining urine specimen when determining albumen and its expression using mass spectrometry method
Including digestion step.In specific embodiments, with the albumen in protease digestion urine specimen.
In specific embodiments, the mass spectrometry method is more reaction monitorings.Especially by the label peptide fragment of protein,
The good mother and sons' ion pair of mass spectrum response signal is screened, the list of mother and sons' ion pair is constructed, optimizes Mass Spectrometer Method collision energy and electricity
The method of pressure is determined to for carrying out qualitative and quantitative detecting analysis mother and sons' ion pair list and testing conditions.
Specifically, labelled peptide, the SEQ of Antithrombin Ⅲ shown in SEQ ID NO:1 and/or SEQ ID NO:2 are detected
α -1- acidoglycoprotein 1 shown in the labelled peptide of Complement C_3 shown in ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5
Labelled peptide, the labelled peptide of serum transferrin shown in SEQ ID NO:6, Ctsz shown in SEQ ID NO:7
Labelled peptide, and combinations thereof.
Based on the quantitative detecting method of the above-mentioned five kinds of albumen of urine, this five kinds can be carried out in crowd with it with combined standard product
The foundation of albumen baseline, can the content range based on normal person provide auxiliary evaluation method to myocardial infarction diagnosis, and can be with
Monitor progression of disease and therapeutic effect.
According to some embodiments, a kind of method for monitoring the severity of subject's myocardial infarction is provided,
Comprising steps of
1) urine specimen of subject and normal healthy controls are obtained,
2) it is compared with normal healthy controls, determines the expression of albumen selected from the following in subject's urine specimen: anticoagulation
Enzyme-III, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, Ctsz, and combinations thereof,
3) expression of albumen described in the expression of albumen described in subject and normal healthy controls is compared,
4) determine whether the myocardial infarction of the subject is in progress.
According to some embodiments, a kind of method for evaluating subject's myocardial infarction therapeutic effect is provided, is wrapped
Include step:
1) urine specimen before acquisition subject and after treatment,
2) expression of albumen selected from the following in the urine specimen before subject and after treatment: anticoagulation is determined
Enzyme-III, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, Ctsz, and combinations thereof,
3) expression of the albumen and the expression of the albumen after treatment are compared before treating,
4) the myocardial infarction treatment validity of the subject is determined.
Detailed description of the invention
The MRM ion pair map of Figure 1A to Fig. 1 E: five kinds of protein specificity peptide fragments.Wherein:
Figure 1A are as follows: the ion pair map of Antithrombin Ⅲ (ANT3) label peptide fragment DDLYVSDAFHK；
Figure 1B are as follows: the ion pair map of Complement C_3 (CO3) label peptide fragment DFDFVPPVVR；
Fig. 1 C are as follows: the ion pair map of α -1- acidoglycoprotein 1 (A1AG1) label peptide fragment SDVVYTDWK；
Fig. 1 D are as follows: the ion pair map of serum transferrin (TRFE) label peptide fragment DGAGDVAFVK；
Fig. 1 E are as follows: the ion pair map of Ctsz (CATZ) label peptide fragment VGDYGSLSGR.
The accuracy of Fig. 2A to Fig. 2 D:MRM analysis.Wherein:
Fig. 2A and Fig. 2 B is respectively the same egg of Antithrombin Ⅲ (ANT3) and α -1- acidoglycoprotein 1 (A1AG1)
White two peptides have similar quantitative trend between three groups；Fig. 2 C and Fig. 2 D are respectively A1AG (R2=0.9509) and ANT3 (R2=
0.8201) two peptides of same albumen quantitative result consistency figure in different samples, wherein MI represents myocardial infarction.
Fig. 3 A to Fig. 3 E: protein Antithrombin Ⅲ (ANT3, Fig. 3 A), Complement C_3 (CO3, Fig. 3 B), α -1- acid sugar
Albumen 1 (A1AG1, Fig. 3 C), serum transferrin (TRFE, Fig. 3 D) and Ctsz (CATZ, Fig. 3 E) in control group and
The MRM testing result of myocardial infarction group (before treatment and after treatment).C is Normal group, and M is T before the treatment of myocardial infarction group
After the treatment of myocardial infarction group.
The ROC curve of Fig. 4: five kinds of protein combinations.
Specific embodiment
Below in conjunction with attached drawing, the present invention is further illustrated by embodiment, but not as limitation of the present invention.It mentions below
Specific material and its source used in embodiment of the present invention are supplied.However, it should be understood that these are only example
Property, it is not intended to the limitation present invention, it is same or similar with the type of following reagent and instrument, model, quality, property or function
Material may be incorporated for implement the present invention.Experimental method used in following embodiments is routine unless otherwise specified
Method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1: urine myocardial infarction GAP-associated protein GAP quality detection
We use isotope labelling opposite and the method for absolute quantitation (iTRAQ) marked difference proteomics is in urine
Middle screening myocardial infarction related protein.
Material and reagent
1) instrument: 5600 mass spectrograph of Triple TOF (Absciex company).
2) main agents: trypsase (Promega company)；(3CC, 60mg, Waters are public for C18 solid phase extraction column
Department)；4 marks iTRAQ reagent (Absciex company)；C18 reverse-phase chromatographic column (4.6mm × 250mm, C18,3 μm, Waters company).
3) sample: the urine and 7 normal controls of 7 myocardial infarction patients (in morbidity 12 hours, and seven days after treatment)
The urine of group comes from the 4th affiliated hospital, Jilin University.
The preparation of 1.1 urine proteins
Take urine sample under the conditions of 4 DEG C with 3,500g centrifugation 15min removal cell fragment.Supernatant is taken, is added with 1:3 ratio
Enter the dehydrated alcohol (- 20 DEG C) of pre-cooling, places 2h protein precipitation under the conditions of 4 DEG C.With 4 DEG C of centrifugation 30min of revolving speed of 12,000g
After take protein precipitation fractions, and it is clean that dehydrated alcohol volatilized.Protein lysate is added, liquid-transfering gun is blown and beaten repeatedly, fills albumen
Division solution.By the solution of redissolution with 4 DEG C of centrifugation 30min of revolving speed of 12,000g, removal precipitating retains albumen supernatant.Gained albumen
Quality sample measures protein concentration with Bradford method.By mass mixings such as protein samples, -80 DEG C freeze it is spare.
1.2 filter membrane auxilin digestions
Take 10KD filtration pipe, be added 200 μ L UA (8M urea+0.1M Tris, pH 8.5) to film on, be put into hypervelocity from
Scheming, 14,000g, 18 DEG C of revolving speed, centrifugation 5min discards lower layer's waste liquid, is repeated twice to liquid under.Everyone such as takes at the matter
Protein example mixing is measured, adds in filtration pipe, 14 000g, 18 DEG C, is centrifuged about 20min.Discard lower layer's waste liquid.It is managed to filtration
200 μ L UA of middle addition, be vortexed concussion 1min, is put into ultracentrifuge, and 14 000g, are centrifuged about 30min by 18 DEG C.Discard lower layer
Waste liquid.It is repeated twice.200 μ L 20mM DTT are added in filtration pipe, be vortexed concussion 1min, 37 DEG C of water bath with thermostatic control 1h.Water-bath terminates
Sample is taken out afterwards, 14 000g, 18 DEG C, is centrifuged about 10min, discards lower layer's waste liquid.200 μ L UA, whirlpool is added into filtration pipe
Rotation concussion 1min, 14 000g, 18 DEG C, centrifugation about 10min discards lower layer's waste liquid, is repeated twice to liquid under.It is managed to filtration
200 μ L 50mM IAA of middle addition, be vortexed concussion 1min, is protected from light, room temperature, reacts 45min.After reaction, by sample 14
000g, is centrifuged about 10min, discards lower layer's waste liquid by 18 DEG C.200 μ L UA are added into filtration pipe, be vortexed concussion 1min, and 14
000g, is centrifuged about 30min, discards lower layer's waste liquid by 18 DEG C.It is repeated twice.200 μ L 25mM NH are added into filtration pipe4HCO3,
Be vortexed concussion 1min, is put into ultracentrifuge, and 14 000g, are centrifuged about 30min, discard lower layer's waste liquid by 18 DEG C.It is repeated twice.To
200 μ L 25mM NH are added in filtration pipe4HCO3, be vortexed concussion 1min.In the ratio of trypsase and protein quality ratio 1:50
Trypsase is added, the concussion that is vortexed mixes.Sample is fixed in water drift, is put into the beaker for filling 1L ice water, is put into microwave
Furnace, Gao Huo, microwave 1min.37 degree of overnight incubations.Sample is put into ultracentrifuge, and 14 000g 18 DEG C, are centrifuged about 10min, receive
Collect lower layer's enzymolysis sample.
1.3C18 Solid Phase Extraction
C18 solid-phase extraction column is taken out, solid-phase extraction device is placed in.500 μ L, 100% acetonitrile is added into pillar and carries out C18
Activation, drip net, be repeated twice naturally to liquid.500 μ L, 1 ‰ formic acid is added into pillar, cleans pillar, is repeated twice.Add
Enter the good enzymolysis liquid of digestion, is dripped naturally to liquid net.500 μ L, 1 ‰ formic acid is added into pillar and carries out cleaning pillar desalination, weight
It is four times multiple.The peptide fragment that 500 μ L, 100% acetonitrile is purified by flash is added into pillar, drips naturally to liquid, is collected and is dripped with EP pipe
Eluent, vacuum drains.It is molten with 1 ‰ formic acid weight to drain rear sample, measures peptide concentration with BCA method, 100ug peptide fragment is taken to carry out
Subsequent experimental.
1.4iTRAQ marked difference Proteomic analysis
By Normal group, myocardial infarction group and myocardial infarction treatment group polypeptide sample mixed in equal amounts prepare hybrid standard
Product.Hybrid standard product, Normal group, myocardial infarction group and myocardial infarction treatment group use 114,115,116 and 117 4 marks respectively
ITRAQ quantitative reagent label.By each component sample (disease group and control group) mixed in equal amounts after label.And carry out offline high pH
High performance liquid chromatography separation, the eluent of collection are placed in rotatory vacuum drying instrument, vacuum drain after redissolve in 1 ‰ formic acid into
Row LC-MS/MS analysis.Each sample carries out mass spectrum three times and repeats.Gained mass spectrogram is by Mascot and Scaffold software point
Analysis, database are Swissprot human protein database (www.uniprot.org), screen differential protein.Albumen is set
1%FDR, polypeptide 1%FDR, the more than two special peptide fragments of each Identification of Fusion Protein are screening criteria, identify albumen number 2086 altogether
It is a.Standard density Discrete point analysis indicates reproducible (the average R of experimental technique2=0.89).With 2 times or more variation and p <
0.05 is used as screening conditions, has 175 and 71 albumen to change in myocardial infarction group and treatment group's expression quantity respectively.
Embodiment 2: urine protein Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin and
The Mass Spectrometer Method of Ctsz
To carry out these Differential proteomic analysis results better further belowly in further more larger scale clinical sample
Application detection, we supervise the differential protein that identifies using targeting mass spectrometric analysis method to differential protein
Control.More reaction monitorings (Multiple Reaction Monitor, MRM) are a kind of target mass spectrums based on second order ms signal
Quantitative analysis tech assists without special chemical labeling, can carry out absolute and relative quantitative determination simultaneously.
Material and reagent
1) instrument: 6500 mass spectrograph of QTRAP (AB Sciex company).
2) main agents: trypsase (Promega company)；(3CC, 60mg, Waters are public for C18 solid phase extraction column
Department).
3) sample: the urine of 15 myocardial infarction patients (in morbidity 12 hours, and seven days after treatment) and 15 it is normal right
According to the urine of group, the 4th affiliated hospital, Jilin University is come from.
Mother and sons' ion pair list of 2.1 differential proteins
Based on Given information or assume information setting Mass Spectrometer Method rule, signal record carried out to legal ion,
Removal is not largely inconsistent the interference of normally ion signal, to obtain a kind of data acquiring mode of Information in Mass Spectra.Specifically,
It is to select parent-daughter ion pair according to polypeptide parent ion mass number and fragment ion masses number, permit compliance with the mother of setting
Ion enters collision cell, and after the completion of collision, only record sets daughter ion signal.By the selection twice of parent ion and daughter ion,
Interfering ion is removed, Chemical Background is reduced, to improve sensitivity.
Differential protein carries out the matching analysis of target mass spectrogram and urine protein database spectrogram, has formulated target mass spectrum
Mother and sons' ion pair of detection.Protein digestion and peptide fragment purification process are same as above, and wherein MRM testing result is consistent with iTRAQ result
11 kinds of protein, mother and sons' ion pair of feature peptide fragment is listed as follows (table 1):
Wherein, MS represents mass spectrum；MS/MS represents second order ms；Cluster voltage is removed in DP representative；CE represents collision energy.
2.2 Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin and Ctsz MRM
Testing result and ROC curve analysis
By literature search, our 5 kinds of albumen of new discovery may be used as potential myocardial infarction marker, before having application
Scape, including antiserum transferrins, fibrin ferment-III, Complement C_3, α -1- acidoglycoprotein 1 and Ctsz.We select
Antithrombin Ⅲ is selected, two polypeptides of α -1- acidoglycoprotein 1 carry out MRM quantitative analysis and Complement C_3, serum turn iron egg
White and Ctsz a polypeptide carries out MRM quantitative analysis.By comparing two polypeptides of the same albumen, Wo Menfa
Existing two protein quantification results are consistent, and their R2 is all larger than 0.8.The accuracy that result above prompts MRM quantitative
(Fig. 2A to Fig. 2 D).Quantitative analysis is carried out to these polypeptides, it has been found that these protein expressions are equal in myocardial infarction group
Obvious up-regulation, and restored (be shown in Table 2 and Fig. 3 A to Fig. 3 E) after the treatment.
The quantitative result of 2 five kinds of albumen of table
MI: myocardial infarction；The combination of a: five albumen is early diagnosed；B: five kinds of albumen restore just after the treatment
Often；*:p<0.05；**:p<0.01；* *: p < 0.001 (can pass through UniProt database (http://www.uniprot.org/
Uniprot/? query=reviewed%3Ayes retrieves protein sequence number, obtains Protein Information).
In turn, we carry out further recipient's operating characteristic curve (receiver operating to it
Characteristic, ROC) tracing analysis, thus judge this five kinds of protein to distinguish whether the ability of myocardial infarction.Its from
Son is shown in Figure 1A to Fig. 1 E to spectrogram.Wherein the feature peptide section sequence of Complement C_3 curve is DFDFVPPVVR, α -1- acidoglycoprotein 1
Feature peptide section sequence be SDVVYTDWK.The label peptide section sequence of serum transferrin is DGAGDVAFVK, and serum turns iron egg
White feature peptide section sequence is DGAGDVAFVK, and the feature peptide section sequence of Ctsz is VGDYGSLSGR, the results show that
The ROC area under the curve AUC that this five kinds of albumen are used alone is all larger than 0.8.In turn, we are by five kinds of spies of this five kinds of albumen
The result for levying peptide fragment carries out confluence analysis.Our integrated results show that AUC analysis result is 0.95, specificity 84%, susceptibility
93% (being shown in Table 3 and Fig. 4).Quantitative result and feature polypeptide ROC curve the analysis result of remaining 6 kinds of albumen are referring to table 4 and table 5.
Additionally, it has been found that the expression quantity of above-mentioned five kinds of albumen after myocardial infarction treatment relative to treatment before dropped
It is low, prompt these albumen there are the potentiality for monitoring progression of disease and therapeutic effect.These parameters show this five kinds of protein pair
In the diagnosis of myocardial infarction, the state of an illness and treatment detection etc. have preferable potential applicability in clinical practice.
The feature polypeptide ROC curve of 3 five kinds of albumen of table analyzes result
AUC: area under the curve.
The quantitative result of remaining the 6 kinds of albumen of table 4
Wherein, *: p < 0.05；**:p<0.01.
The feature polypeptide ROC curve of 5 remaining albumen of table analyzes result
AUC: area under the curve.
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Sequence table
<110>Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
<120>protein marker of urine myocardial infarction and its purposes in diagnosis and prognosis
<130> 370009CG
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of Antithrombin Ⅲ
<400> 1
Leu Pro Gly Ile Val Ala Glu Gly Arg
1 5
<210> 2
<211> 11
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of Antithrombin Ⅲ
<400> 2
Asp Asp Leu Tyr Val Ser Asp Ala Phe His Lys
1 5 10
<210> 3
<211> 10
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of Complement C_3
<400> 3
Asp Phe Asp Phe Val Pro Pro Val Val Arg
1 5 10
<210> 4
<211> 8
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of α -1- acidoglycoprotein 1
<400> 4
Thr Glu Asp Thr Ile Phe Leu Arg
1 5
<210> 5
<211> 9
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of α -1- acidoglycoprotein 1
<400> 5
Ser Asp Val Val Tyr Thr Asp Trp Lys
1 5
<210> 6
<211> 10
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of serum transferrin
<400> 6
Asp Gly Ala Gly Asp Val Ala Phe Val Lys
1 5 10
<210> 7
<211> 10
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of Ctsz
<400> 7
Val Gly Asp Tyr Gly Ser Leu Ser Gly Arg
1 5 10
<210> 8
<211> 8
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of albumin
<400> 8
Asp Leu Gly Glu Glu Asn Phe Lys
1 5
<210> 9
<211> 10
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of albumin
<400> 9
Leu Val Asn Glu Val Thr Glu Phe Ala Lys
1 5 10
<210> 10
<211> 16
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of Annexin A1
<400> 10
Gly Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg
1 5 10 15
<210> 11
<211> 9
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of neutrophil leucocyte alexin 1
<400> 11
Ile Pro Ala Cys Ile Ala Gly Glu Arg
1 5
<210> 12
<211> 12
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of interior acrasin 1
<400> 12
Glu Trp Thr Cys Ser Ser Ser Pro Ser Leu Pro Arg
1 5 10
<210> 13
<211> 8
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of slightly rich proline protein 3
<400> 13
Val Pro Glu Pro Gly Tyr Thr Lys
1 5
<210> 14
<211> 8
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of Delta albumen homology albumen 1
<400> 14
Cys Pro Ala Gly Phe Ile Asp Lys
1 5
<210> 15
<211> 8
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of slightly rich proline protein 3
<400> 15
Val Pro Val Pro Gly Tyr Thr Lys
1 5
Claims (14)
- The combination of the indentifying substance of albumen below 1. preparation for diagnose and/or the reagent of the myocardial infarction of prognosis subject in Purposes: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, Ctsz.
- 2. purposes according to claim 1, wherein the diagnosis is early diagnosis.
- 3. purposes according to claim 2, wherein the expression of albumen below improves instruction compared to normal healthy controls The subject suffers from myocardial infarction: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, tissue Protease Z, and combinations thereof.
- 4. purposes according to claim 1, wherein the prognosis refers to the curative effect evaluation of the myocardial infarction of subject.
- 5. purposes according to claim 4, wherein the expression of albumen below reduces instruction before compared to treatment The validity of the myocardial infarction treatment of the subject: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum turn iron Albumen, Ctsz, and combinations thereof.
- 6. purposes according to any one of claim 1 to 5, wherein the indentifying substance is Mass Spectrometric Identification reagent, antibody Or its antigen-binding fragment.
- 7. purposes according to claim 6, wherein the antibody is monoclonal antibody.
- 8. purposes according to claim 6, wherein the Mass Spectrometric Identification reagent is used in more reaction monitorings.
- 9. purposes according to claim 8, wherein the Mass Spectrometric Identification reagent are as follows: SEQ ID NO:1 and/or SEQ ID The mirror of the labelled peptide of Complement C_3 shown in the indentifying substance of the labelled peptide of Antithrombin Ⅲ shown in NO:2, SEQ ID NO:3 Determine indentifying substance, the SEQ of the labelled peptide of α -1- acidoglycoprotein 1 shown in reagent, SEQ ID NO:4 and/or SEQ ID NO:5 The mark of Ctsz shown in the indentifying substance of the labelled peptide of serum transferrin shown in ID NO:6, SEQ ID NO:7 The indentifying substance of label peptide, and combinations thereof.
- 10. the purposes according to claim 3 or 5, wherein the expression is in the urine specimen of the subject Measurement.
- 11. purposes according to claim 10, wherein the subject is people.
- 12. a kind of for diagnosing and/or the kit or chip of prognosis myocardial infarction, it includes the identification of albumen below examinations Agent: Antithrombin Ⅲ, Complement C_3, α -1- acidoglycoprotein 1, serum transferrin, Ctsz；The indentifying substance is Mass Spectrometric Identification reagent, antibody or its antigen-binding fragment；The diagnosis and/or prognosis are selected from: the early diagnosis of myocardial infarction, the assessment of curative effect, the myocardial infarction for monitoring subject Severity, and combinations thereof.
- 13. according to claim 12 for diagnosing and/or the kit or chip of prognosis myocardial infarction, wherein described Antibody is monoclonal antibody.
- 14. according to claim 12 for diagnosing and/or the kit or chip of prognosis myocardial infarction, wherein described Kit or chip also include the indentifying substance of albumen selected from the following:Albumin, Annexin A1, neutrophil leucocyte alexin 1, interior acrasin 1, slightly rich proline protein 3, Delta albumen are same Source protein 1, and combinations thereof.
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| CN108663448B (en) * | 2018-05-14 | 2021-07-13 | 中国医学科学院医药生物技术研究所 | Method for detecting related substances in compound amino acid injection |
| CN108753957A (en) * | 2018-07-03 | 2018-11-06 | 北京泱深生物信息技术有限公司 | The IVD genes of diagnosing myocardial infarction and its application |
| CN111896651B (en) * | 2020-07-30 | 2021-04-23 | 山东大学 | Agkistrodon halys venom thrombin-like enzyme characteristic polypeptide and application thereof |
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| CN1751128A (en) * | 2002-12-24 | 2006-03-22 | 博适公司 | Markers for differential diagnosis and methods of use thereof |
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|---|---|---|---|---|
| CN1751128A (en) * | 2002-12-24 | 2006-03-22 | 博适公司 | Markers for differential diagnosis and methods of use thereof |
| CN102812359A (en) * | 2009-11-13 | 2012-12-05 | Bg医药公司 | Risk Factors And Prediction Of Myocardial Infarction |
| CN105223348A (en) * | 2014-05-26 | 2016-01-06 | 北京九强生物技术股份有限公司 | The latex enhancing immune turbidimetry detection kit of people's Antithrombin III |
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