CN110286234A - The protein markers of gestational diabetes mellitus and its purposes in early diagnosis in urine - Google Patents
The protein markers of gestational diabetes mellitus and its purposes in early diagnosis in urine Download PDFInfo
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Abstract
Purposes the present invention relates to the protein markers of gestational diabetes mellitus in urine and its in early diagnosis.Specifically, the present invention relates to purposes of the indentifying substance of protein selected from the following in the reagent that preparation is used for glycosuria gravidarum disease early diagnosis: nov protein homologue (Protein NOV homolog), (Peroxiredoxin-5 of peroxiredoxin -5, PRDX-5), hepatocyte growth factor-like protein (Hepatocyte growth factor-like protein, HFGL) and combinations thereof.These three protein Ns OV albumen homology object, peroxiredoxin -5 and hepatocyte growth factor-like protein and combinations thereof have preferable potential applicability in clinical practice for early diagnosis of gestational diabetes mellitus etc..
Description
Invention field
The present invention relates to field of biotechnology, relate more specifically to gestational diabetes mellitus in Samples subjects, such as urine
Protein markers and its purposes in early diagnosis.
Background of invention
Gestational diabetes (Gestational diabetesmellitus, GDM), which refer to, to be occurred in the pregnancy period or sends out for the first time
Existing impaired glucose tolerance.According to different crowd and diagnostic criteria, the disease incidence highest of GDM up to 18%, and with other ethnic phases
Than asian ancestry women is easier that GDM occurs in the gestational period.GDM has adverse effect to the health of fetus and pregnant woman, may cause tire
Stop educating, premature labor, difficult labour, infection, macrosomia, icterus neonatorum, neonatal hypoglycemia, hyperbilirubinemia, pre-eclampsia etc..
The risk that GDM patient and its fetus suffer from diabetes, obesity and metabolic syndrome in the future dramatically increases, and not only increases illness family
Living burden, and greatly increase national medical insurance expenditure.Therefore, EARLY RECOGNITION GDM vulnerable crowd is particularly significant, so as to the greatest extent
Early strict control risk factor prevents the appearance of hyperglycemia state, reduces Averse pregnancy outcomes and suffers from metabolic disease in the future
Risk.Meanwhile GDM is also the alert status of diabetes B, early prediction index is necessarily beneficial to diabetes B
Prediction.
Current more generally accepted GDM diagnostic method be in pregnant 24 weeks to 28 weeks progress 75g oral glucose tolerance tests, it is bright
True Diagnostic Time relatively lags behind, and the marker of GDM crowd will occur if there is pregnant preceding or pregnant early stage Accurate Prediction, clinical early
Phase intervenes just more targetedly.Many researchers were once ground using the classical way including Enzyme Linked Immunoadsorbent Assay
Study carefully, filters out the GDM of the indexs such as adiponectin, free fatty acid, sex hormone binding globulin and placental growth hormone alternately
Early diagnosis marker.But due to these index poor specificities, and only to be screened from the index of known maturation, range is small,
Its sensibility and specificity needs to be considered, and does not gain public acceptance.Therefore, using advanced protein research technology directly in albumen
Level explore it is new, more preferably, clinical more practical GDM early diagnosis marker be very necessary.
Currently, many laboratories utilize proteomic techniques both at home and abroad, the difference of gestational diabetes mellitus is detected in blood plasma
Expression.Suo Mingli in 2009 et al. is applied to the research of GDM using proteomics method for the first time.Research application SELDI-
TOF-MS technology detects and depicts the serum proteins spectrogram of 30 GDM patients and 30 normal pregnancies, it was found that mass-to-charge ratio
It is expressed for 2868.9,3448.3,5477.7,4695.5 and 4786.7 protein matter in two group differences.In recent years increasingly
More laboratories utilize SELDI-TOF technology, and LC-MS quantitative technique, iTRAQ quantitative technique, TMT quantitative technique are in plasma proteins
Gestational diabetes mellitus marker is identified in group.C reactive protein, sex hormone binding globulin, the special beta-1 sugar of Ficolin3, gestation
Albumen 4, lipoprotein E, coagulation factor 9, fibrinogen α chain, Insulin-like Growth hormonebinding protein 5 etc. are a series of potential pregnant
Diabetes of being pregnent marker is found and verifies.
Therefore, quick, the sensitive and effective method of detection gestational diabetes mellitus is badly in need of in this field.
Summary of the invention
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 gestational diabetes mellitus related protein.
Targeting analysis (parallel reaction) monitoring (Parallel Reaction Monitoring, PRM) is a kind of based on two
The target mass spectrum quantitative analysis tech of grade mass signal, compared to traditional Selective reaction monitoring (SelectedReaction
Monitoring, SRM) technology, without being pre-designed parent ion/daughter ion unpaired message of targeting protein, saving experiment is set
Meter and operating time；And selectivity is higher, more preferably, reproducibility is more preferable for sensitivity, and the anti-interference ability in complex background is stronger.
It is not necessarily to commercial antibody, overcomes the limitation based on immunization method to antibody specificity and titre.Targeting analysis is (parallel anti-
Answer) monitoring technology can simultaneously to multiple proteins carry out qualitative and quantitative analysis.
In this application, we collect the urine of 15 gestational diabetes mellitus early stages and the urine (gestation of 15 normal pregnancies
6-10 weeks), quantitative analysis is carried out to urine protein group using novel DIA label-free technology.By being supervised to OPLSDA
Clustering, fold differences analysis and T check analysis, select 48 potential biomarkers, develop based on targeting mass spectrum
Detection technique is simultaneously to nov protein homologue (Protein NOV homolog), hepatocyte growth factor-like protein
The method that protein such as (Hepatocyte growth factor-like protein) are detected, to 15 gestation of collection
The urine of diabetes early stage and the urine (pregnant 6-10 weeks) of 15 normal pregnancies are analyzed, and utilize MASS SPECTRAL DATA ANALYSIS knot
Fruit.
We have found that nov protein homologue (Protein NOV homolog), peroxiredoxin -5
(Peroxiredoxin-5, PRDX-5), hepatocyte growth factor-like protein (Hepatocyte growth factor-like
Protein, HFGL) targeting mass spectrometric data have well to above-mentioned 15 gestational diabetes mellitus groups and 15 normal pregnancy control groups
Discrimination, can quickly, accurately and effectively diagnose gestational diabetes mellitus, and performance when a combination thereof uses is especially prominent.Detect this three
Kind biomarker can be applied to the early diagnosis monitoring of gestational diabetes mellitus, have good potential applicability in clinical practice.
Brief description
The MRM ion pair map of Figure 1A to Fig. 1 C: three kinds of protein specificity peptide fragments.Wherein:
Figure 1A are as follows: the ion pair map of nov protein homologue label peptide fragment AVLDGCSCCLVCAR；
Figure 1B are as follows: the ion pair map of peroxiredoxin -5 (PRDX-5) label peptide fragment LLADPTGAFGK；
Fig. 1 C are as follows: the ion pair map of the labelled peptide SPLNDFQVLR of hepatocyte growth factor-like protein (HFGL)；
The accuracy of Fig. 2A to Fig. 2 B:MRM analysis, in which:
Fig. 2A and Fig. 2 B is respectively nov protein homologue (R2=0.8128) two peptides of the same albumen are in different samples
Middle quantitative result consistency figure.
Fig. 3 A to Fig. 3 C:NOV albumen homology object (Fig. 3 A), peroxiredoxin -5 (PRDX-5, Fig. 3 B), cell growth
Because increment albumen (HFGL, Fig. 3 C) is in the MRM testing result of normal pregnancy group and gestational diabetes mellitus group (before treatment and after treatment).
CON is normal pregnancy group, and GDM is glycosuria gravidarum foot disease.
Fig. 4: in Fig. 4 A to Fig. 4 D, it is bent that Fig. 4 A represents the ROC that nov protein homologue and peroxiredoxin -5 combine
Line, Fig. 4 B represent the combination ROC curve of nov protein homologue and hepatocyte growth factor-like protein, and Fig. 4 C represents peroxide
The combined ROC curve (Fig. 4 C) and Fig. 4 D of reductase -5 and hepatocyte growth factor-like protein represents three kinds of protein combinations
ROC curve.
Detailed description of the invention
In view of the demand of quick, the sensitive and effective method of the urgent need detection gestational diabetes mellitus of this field, on the one hand,
It is selected from the following any one or more this application provides Mass Spectrometric Identification reagent:
1) indentifying substance of the labelled peptide of nov protein homologue shown in SEQ ID NO:1；
2) indentifying substance of the labelled peptide of NOV shown in SEQ ID NO:2；
3) indentifying substance of the labelled peptide of peroxiredoxin -5 (PRDX-5) shown in SEQ ID NO:3；
4) indentifying substance of the labelled peptide of hepatocyte growth factor-like protein (HFGL) shown in SEQ ID NO:4.
On the other hand, this application provides indentifying substances to prepare the purposes in composition, and the composition is for diagnosing
The method of the gestational diabetes mellitus of subject, the indentifying substance are selected from the following any one or more:
1) it is used for the indentifying substance of nov protein homologue；
2) it is used for the indentifying substance of peroxiredoxin -5；
3) it is used for the indentifying substance of hepatocyte growth factor-like protein,
The method comprise the steps that
A. sample is obtained from the subject,
B. optionally, protein included in it is separated from sample,
C. indentifying substance is used, kit or chip determine selected from the following any in the sample from the subject
Or the expression of multiple proteins: nov protein homologue, peroxiredoxin -5, hepatocyte growth factor-like protein, it is excellent
Above-mentioned expression is compared by selection of land with normal healthy controls.
In specific embodiments, described to diagnose the early diagnosis for referring to gestational diabetes mellitus.The early diagnosis refers to:
Gestational diabetes mellitus diagnosis in pregnant 6-10 weeks.
In one embodiment, the use in composition is used to prepare on the way or in diagnosis subject in above-mentioned indentifying substance
Gestational diabetes mellitus method in, any one of selected from the following in the sample from the subject or multinomial instruction is described tested
Person suffers from gestational diabetes mellitus:
1) compared to normal healthy controls, the expression of nov protein homologue is increased；
2) compared to normal healthy controls, hepatocyte growth factor-like protein expression is increased；
3) compared to normal healthy controls, the expression of peroxiredoxin -5 is reduced.
Indentifying substance suitable for the application 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 work
The antibody fragment of property is suitable for the disclosure.
In one embodiment, the expression of protein involved in measuring is surveyed in the urine specimen of subject
Fixed, it is preferable that the subject is people.
In one embodiment, this application involves indentifying substance be used to prepare in composition on the way or examining
In the method for the gestational diabetes mellitus of disconnected subject, using involved in the determination of mass spectrometry method, ELISA method or Western method
The expression of protein.
In a preferred embodiment, this application involves indentifying substance be used to prepare in composition use on the way
Or it in the method for the gestational diabetes mellitus in diagnosis subject, when determining the expression of protein using mass spectrometry method, also wraps
The step of including the protein in sample digestion, preferably using the protein in trypsin digestion sample.
In one embodiment, used in the Mass Spectrometric Identification reagent is monitored in targeting analysis (parallel reaction).
Targeting analysis (parallel reaction) monitoring is a kind of target mass spectrum quantitative analysis tech based on second order ms signal, phase
Compared with traditional Selective reaction monitoring technology, the parent ion/daughter ion unpaired message for being pre-designed targeting protein is not needed, is saved
About experimental design and operating time；And selectivity is higher, more preferably, reproducibility is more preferable for sensitivity, anti-interference in complex background
Ability is stronger.It is no longer limited by commercial antibodies compared to immunization method, overcomes based on immunization method to antibody specificity
With the limitation of titre.Targeting analysis (parallel reaction) monitoring technology can carry out qualitative and quantitative point to multiple proteins simultaneously
Analysis.
This application involves indentifying substance be used to prepare in composition use on the way or diagnosis subject gestation sugar
In the method for urinating disease, when determining the expression of protein using mass spectrometry method, pass through the label peptide fragment of protein, acquisition mark
Label peptide fragment corresponds to all daughter ions of parent ion.It is quantified according to the signal strength of daughter ion.
Here, labelled peptide is the peptide fragment for referring to represent some protein, it is characterised in that exist and specificity is only deposited
In Mr. Yu's protein amino acid sequence.
In one embodiment, this application involves indentifying substance be used to prepare in composition on the way or examining
In the method for the gestational diabetes mellitus of disconnected subject, Mass Spectrometric Identification reagent is selected from the following any one or more:
1) indentifying substance of the labelled peptide of nov protein homologue shown in SEQ ID NO:1；
2) indentifying substance of the labelled peptide of NOV shown in SEQ ID NO:2；
3) indentifying substance of the labelled peptide of peroxiredoxin -5 (PRDX-5) shown in SEQ ID NO:3；
4) indentifying substance of the labelled peptide of hepatocyte growth factor-like protein (HFGL) shown in SEQ ID NO:4.
When using above-mentioned indentifying substance, this application involves indentifying substance be used to prepare the use in composition on the way or
In the method for the gestational diabetes mellitus of diagnosis subject, when determining the expression of protein using mass spectrometry method, pass through inspection
Any one of labelled peptide of protein selected from the following or a variety of expressions to determine protein are surveyed, thus to gestation
Diabetic early diagnoses:
1) labelled peptide of nov protein homologue shown in SEQ ID NO:1；
2) labelled peptide of NOV shown in SEQ ID NO:2；
3) labelled peptide of peroxiredoxin -5 (PRDX-5) shown in SEQ ID NO:3；
4) labelled peptide of hepatocyte growth factor-like protein (HFGL) shown in SEQ ID NO:4.
Another aspect provides a kind of kit or chip for the diagnosis of subject's gestational diabetes mellitus, packets
Containing indentifying substance, the indentifying substance includes any one or more indentifying substances selected from the following:
1) it is used for the indentifying substance of nov protein homologue；
2) it is used for the indentifying substance of peroxiredoxin -5；
3) it is used for the indentifying substance of hepatocyte growth factor-like protein.
In one embodiment, in kit provided by the present application or chip, the gestational diabetes mellitus diagnosis is early
Phase diagnosis.
In one embodiment, in kit provided by the present application or chip, the indentifying substance is Mass Spectrometric Identification
Reagent, antibody or its antigen-binding fragment, wherein the antibody is preferably monoclonal antibody, the antigen-binding fragment is preferably wrapped
It includes but is not limited to: Fab, Fab', (Fab') 2, Fv, ScFv, bispecific antibody, three specific antibodies, four specific antibodies, double-scFv.
Preferably, the Mass Spectrometric Identification reagent is analyzed used in (parallel reaction) monitoring in targeting.
In another embodiment, in kit provided by the present application or chip, the indentifying substance is for described
The urine of subject, and the subject is preferably people.
In a preferred embodiment, in kit provided by the present application or chip, the Mass Spectrometric Identification reagent
It is selected from the following any one or more:
1) indentifying substance of the labelled peptide of nov protein homologue shown in SEQ ID NO:1；
2) indentifying substance of the labelled peptide of NOV shown in SEQ ID NO:2；
3) indentifying substance of the labelled peptide of peroxiredoxin -5 (PRDX-5) shown in SEQ ID NO:3；
4) indentifying substance of the labelled peptide of hepatocyte growth factor-like protein (HFGL) shown in SEQ ID NO:4.
In yet another aspect, present invention also provides kit involved in the application or chip preparation for diagnosing
Purposes in the composition of the gestational diabetes mellitus of subject.Preferably, composition is used to prepare in the kit or chip
With on the way, any one of selected from the following or multinomial instruction subject suffers from glycosuria gravidarum in the sample from the subject
Disease:
1) compared to normal healthy controls, the expression of nov protein homologue is increased；
2) compared to normal healthy controls, hepatocyte growth factor-like protein expression is increased；
3) compared to normal healthy controls, the expression of peroxiredoxin -5 is reduced.
When using above-mentioned indentifying substance, this application involves the kit or chip be used to prepare the use of composition
On the way, when determining the expression of protein using mass spectrometry method, in the labelled peptide by detecting protein selected from the following
It is any or a variety of determine the expression of protein, to be early diagnosed to patients with gestational diabetes:
1) labelled peptide of nov protein homologue shown in SEQ ID NO:1；
2) labelled peptide of NOV shown in SEQ ID NO:2；
3) labelled peptide of peroxiredoxin -5 (PRDX-5) shown in SEQ ID NO:3；
4) labelled peptide of hepatocyte growth factor-like protein (HFGL) shown in SEQ ID NO:4.
Another aspect, the application provide the gestational diabetes mellitus of diagnosis subject, preferably 6-10 weeks glycosuria gravidarum of gestation
The method of disease, the described method comprises the following steps:
A. sample is obtained from the subject,
B. optionally, protein included in it is separated from sample,
C. it is determined using above-mentioned indentifying substance or kit or chip selected from the following in the sample from the subject
The expression of any one or more protein: nov protein homologue, peroxiredoxin -5, hepatocyte growth factor sample
Albumen,
D. the expression and normal healthy controls are compared into the glycosuria gravidarum diseased state to determine subject,
Preferably, wherein any one of selected from the following in the sample from the subject or multinomial instruction subject
With gestational diabetes mellitus:
1) compared to normal healthy controls, the expression of nov protein homologue is increased；
2) compared to normal healthy controls, hepatocyte growth factor-like protein expression is increased；
3) compared to normal healthy controls, the expression of peroxiredoxin -5 is reduced.
Sample based on subject, for example, in urine above-mentioned protein quantitative detecting method, can be used with combined standard product
The foundation of these protein baselines in its carry out crowd, can be based on the content range of normal pregnancy, to patients with gestational diabetes
It is early diagnosed.
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.It is to be understood, however, 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: gestational diabetes mellitus GAP-associated protein GAP quality detection in urine
We are with data dependent/non-dependent acquisition method (Data independent acquisition, DIA) in urine
Screen gestational diabetes mellitus related protein.
Material and reagent
1) instrument: Orbitrap Fusion Lumos Tribrid mass spectrograph (Thermo Scientific company).
2) main agents: trypsase (Promega company)；(3CC, 60mg, Waters are public for C18 solid phase extraction column
Department)；C18 reverse-phase chromatographic column (4.6mm × 250mm, C18,3 μm, Waters company).
3) sample: the urine of 15 patients with gestational diabetes (pregnant 6-10 weeks) and the urine of 15 Normal groups are come
From BJ Union Hospital.
The collection of 1.1 human urine samples and the enrichment of Urine proteins
Empty stomach urina sanguinis is collected, the revolving speed of 5000g is centrifuged 30min, removal precipitating.Supernatant ethanol precipitation, 4 spend night to analyse
Protein out.Albumen precipitation is redissolved with lysate.Using the concentration for the human urine albumen that Bradford method measurement is collected into.Egg
White sample is analyzed with SDS-PAGE.
1.2 proteolysis
Proteolysis is carried out with enzymatic cleavage methods on film.Protein sample first uses 20mM DTT to restore 95 degree of 5min, then uses 50mM
IAA is alkylated (room temperature 45min), and is loaded on 30KD filter membrane, and centrifugation discards lower layer's waste liquid.Protein sample UA solution on film
(urea containing 8M) cleans twice, and is cleaned twice with 25mM ammonium bicarbonate soln.Protein sample is spent with 1:50 pancreatin 37 on film
Night enzymatic hydrolysis, is collected by centrifugation polypeptide after enzymatic hydrolysis.Polypeptide extracts column extracting with C18 after digestion, and vacuum is drained.Drain rear polypeptide BCA
It is quantitative that method carries out polypeptide.
1.3 building libraries
In order to construct spectrogram library, by all urine samples (disease group and control group) mixed in equal amounts.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.The sample of each component data dependency acquisition method (DDA, Data dependent
Acquisition) mode acquires, collected initial data with Proteome Discoverer (Thermo Scientific,
Germany) software retrieval, search condition are as follows: search parameter is as follows: database SwissProt human (under be loaded in
The website Uniprot) digestion mode: trypsase；Allow two mistake enzyme sites；Fixed modification: cysteine alkylation；It can turn revisionism
Decorations: aspartic acid and glutamine deaminizing, methionine oxidation；Lysine and polypeptide N-terminal carbamylation；Parent ion quality is missed
Difference: 20ppm；Product ion mass error: 0.05Da.Protein level FDR < 1%, each albumen at least contain a unique peptide.
Search result imports Spectronaut Pulsar (Biognosys, Switzerland) software and generates database.
1.4DIA analyzes experimental data:
30 samples are analyzed with 1D-LC-MS/MS respectively.Each sample DIA mode carries out data acquisition.The number of DIA acquisition
It is handled according to Spectronaut Pulsar software.The spectrogram library of above-mentioned building is retrieved, search parameter is same as above.Export data knot
Fruit.Using 1.5 times or more variations and p < 0.05 as screening conditions, identify differential protein in gestational diabetes mellitus and normal pregnancy
It changes.
Embodiment 2: urine protein nov protein homologue, peroxiredoxin -5, hepatocyte growth factor-like protein
Mass Spectrometer Method.
For better further belowly to these Differential proteomic analysis results in further more larger scale clinical sample
Using detection, we are monitored the differential protein identified using targeting mass spectrometric analysis method to differential protein.
Targeting analysis (parallel reaction) monitoring is a kind of target mass spectrum quantitative analysis tech based on second order ms signal, compared to tradition
Selective reaction monitoring technology, do not need the parent ion/daughter ion unpaired message for being pre-designed targeting protein, save experiment and set
Meter and operating time；And selectivity is higher, more preferably, reproducibility is more preferable for sensitivity, and the anti-interference ability in complex background is stronger.
It is no longer limited by commercial antibodies compared to immunization method, overcomes based on immunization method to antibody specificity and titre
Limitation.Targeting analysis (parallel reaction) monitoring technology can carry out opposite and absolute quantitation simultaneously.
Material and reagent
1) instrument: Triple TOF5600 mass spectrograph (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 patients with gestational diabetes (pregnant 6-10 weeks) and the urine of 15 Normal groups are come
From BJ Union Hospital.
2.1 sample preparations:
The urine of 15 patients with gestational diabetes (pregnant 6-10 weeks) and the urine of 15 Normal groups, take 4ml respectively
Urine carries out trypsin digestion.Sample carries out peptide concentration measurement with BCA method after enzymatic hydrolysis.Each sample takes 10.5ug
1.5uL iRT standard peptide is added in (0.5ug/uL).And take equivalent sample preparation aggregate sample.
The screening of 2.2PRM polypeptide:
Differential protein to be verified is chosen, PRM verifying is carried out.PRM polypeptide screening is carried out with 3.6 software of Skyline.
First screened with mixed polypeptide.Selection has preferable spectrogram, and the energy in mixing sample in urine protein group database
It is enough identifying or with compared with high s/n ratio peak polypeptide carry out subsequent authentication.
2.3PRM verifying:
PRM analysis is carried out to the polypeptide that can be used for PRM verifying screened in 2.2.Above-mentioned 30 samples are separately verified,
Each sample analyzes polypeptide to be verified with schedule mode.For guarantee the quality of data, all samples loading it
After preceding and loading and between every 8-10 sample, each analysis for carrying out mixed once sample is controlled as quality, is observed whole
In a analytic process, the stability of instrument signal.To guarantee the quality of data, iRT standard peptide analysis, observation are added in each sample
In analytic process, the stability of chromatographic retention.Each sample carries out technology replicate analysis twice.Difference group sample is upset suitable
Sequence is interspersed to be analyzed by mass spectrometry, to reduce systematic error.
The analysis of 2.4PRM data:
PRM data analysis is carried out with 3.6 software of Skyline.All results are imported into Skyline software, hand picking
Correct peak exports all polypeptide results of all samples.+ 2~+5 electricity of each sample are extracted with Progenesis software
The total ion current intensity (TIC) of lotus.The total ion current intensity of the mass spectral results of each polypeptide of each sample samples is equal
One changes, and corrects the error of applied sample amount and mass signal intensity.Quantitative analysis is carried out to each polypeptide result, is screened between different groups
Differential protein, and be compared with DIA result.ROC curve analysis is carried out with Metaboanalyst, filtering out can have
Highly sensitive and specificity disease marker or marker combination.
The result shows that the quantitative result of several albumen is consistent with DIA result, and sensitivity and specificity with higher,
Its feature polypeptide is listed as follows (table 1).
3 kinds of protein characteristic peptide fragment lists in 1 urine of table
By comparing two polypeptides (nov protein homologue) of the same albumen, it has been found that two polypeptide quantitative results
It is consistent, and their R2 is all larger than 0.8.The accuracy (A and 2B referring to fig. 2) that result above prompts MRM quantitative.
We can be used as potential gestational diabetes mellitus marker by three kinds of protein of new discovery, have application prospect, including
It is aobvious for the differentiation of gestational diabetes mellitus for nov protein homologue, peroxiredoxin -5, hepatocyte growth factor-like protein
It writes.Quantitative analysis is carried out to aforementioned polypeptides, it has been found that this nov protein homologue and cell growth factor increment protein expression are pregnant
It is obviously raised in diabetes of being pregnent group, the expression of peroxiredoxin -5 is obviously lowered in gestational diabetes mellitus group (is shown in Table 2 and Fig. 3 A
To Fig. 3 C).
The quantitative result of 2 three kinds of albumen of table
GDM: gestational diabetes mellitus；The combination of a: three albumen is early diagnosed；*:p<0.05；
In turn, we carry out further recipient's operating characteristic curve (receiver operating to it
Characteristic, ROC) tracing analysis, thus judge these three protein distinguish whether the ability of gestational diabetes mellitus.Its from
Son is shown in Figure 1A to Fig. 1 C to spectrogram.Wherein, the feature peptide section sequence of nov protein homologue is AVLDGCSCCLVCAR, peroxidating
The characteristic sequence of object reductase -5 is LLADPTGAFGK, and the characteristic sequence of hepatocyte growth factor-like protein is SPLNDFQVLR.
The results show that area AUC is all larger than 0.7 under the ROC curve that this 3 kinds of protein are used alone.(table 3)
The feature polypeptide ROC curve of 3. 3 kinds of protein of table analyzes result
AUC: area under the curve.
In turn, any two or three of result of three kinds of feature peptide fragments of these three albumen is carried out confluence analysis by us
(Fig. 4).The AUC analysis result that our integrated results show that nov protein homologue and peroxiredoxin -5 combine is 0.851
The combined AUC analysis result of (Fig. 4 A), nov protein homologue and hepatocyte growth factor-like protein is 0.754 (Fig. 4 B), mistake
Oxidoreductase -5 and the combined AUC analysis result of hepatocyte growth factor-like protein are 0.861 (Fig. 4 C), three kinds of albumen
Combined AUC analysis result be AUC analysis result be 0.896 (Fig. 4 D).
In short, our result indicate that, nov protein homologue (Protein NOV homolog), peroxide reduction
Enzyme -5 (Peroxiredoxin-5, PRDX-5), hepatocyte growth factor-like protein (Hepatocyte growth factor-
Like protein, HFGL) there is good discrimination to gestational diabetes mellitus and normal pregnancy control, two of them nov protein is same
Source object, peroxiredoxin -5 and hepatocyte growth factor-like protein, can quickly, accurately and effectively diagnose gestational diabetes mellitus,
And relative to exclusive use, performance when being applied in combination is more prominent.These three biomarkers can be applied to glycosuria gravidarum
The early diagnosis monitoring of disease, has good potential applicability in clinical practice.
Sequence table
<110>Antide and (Hangzhou) medical science and technology Co., Ltd
<120>protein markers of gestational diabetes mellitus and its purposes in early diagnosis in urine
<130> C18P3010
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 14
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of NOV homologue (Protein NOV homolog)
<400> 1
Ala Val Leu Asp Gly Cys Ser Cys Cys Leu Val Cys Ala Arg
1 5 10
<210> 2
<211> 16
<212> PRT
<213>artificial sequence
<220>
<223>labelled peptide of nov protein homologue
<400> 2
Cys Gln Leu Asp Val Leu Leu Pro Glu Pro Asn Cys Pro Ala Pro Arg
1 5 10 15
<210> 3
<211> 11
<212> PRT
<213>artificial sequence
<220>
<223>peroxiredoxin -5(Peroxiredoxin-5, PRDX-5) labelled peptide
<400> 3
Leu Leu Ala Asp Pro Thr Gly Ala Phe Gly Lys
1 5 10
<210> 4
<211> 10
<212> PRT
<213>artificial sequence
<220>
<223>hepatocyte growth factor-like protein (Hepatocyte growth factor-like
Protein, HFGL) labelled peptide
<400> 4
Ser Pro Leu Asn Asp Phe Gln Val Leu Arg
1 5 10
Claims (10)
1. Mass Spectrometric Identification reagent, selected from the following any one or more:
1) the identification examination of the labelled peptide of albumen nov protein homologue shown in SEQ ID NO:1 (Protein NOV homolog)
Agent；
2) indentifying substance of the labelled peptide of NOV shown in SEQ ID NO:2；
3) identification of the labelled peptide of peroxiredoxin -5 (Peroxiredoxin-5, PRDX-5) shown in SEQ ID NO:3
Reagent；
4) (the Hepatocyte growth factor-like of hepatocyte growth factor-like protein shown in SEQ ID NO:4
Protein, HFGL) labelled peptide indentifying substance.
2. indentifying substance is preparing the purposes in composition, the composition is for diagnosing the gestational diabetes mellitus of subject, preferably
Early pregnancy diabetes, i.e., gestation 6-10 weeks gestational diabetes mellitus method, the indentifying substance it is selected from the following any or
It is a variety of:
1) it is used for the indentifying substance of nov protein homologue；
2) it is used for the indentifying substance of peroxiredoxin -5；
3) it is used for the indentifying substance of hepatocyte growth factor-like protein；
It is preferred that wherein the indentifying substance is Mass Spectrometric Identification reagent, preferably the Mass Spectrometric Identification reagent, antibody of claim 1 or
Its antigen-binding fragment, wherein the antibody is preferably monoclonal antibody, the antigen-binding fragment is preferably included, but is not limited to:
Fab, Fab', (Fab') 2, Fv, ScFv, bispecific antibody, three specific antibodies, four specific antibodies, double-scFv.
3. purposes according to claim 2, wherein any one of selected from the following or multinomial instruction subject is with pregnant
It is pregnent diabetes:
1) compared to normal healthy controls, the expression of nov protein homologue is increased；
2) compared to normal healthy controls, hepatocyte growth factor-like protein expression is increased；
3) compared to normal healthy controls, the expression of peroxiredoxin -5 is reduced.
4. purposes according to claim 2 or 3, including using mass spectrometry method, ELISA method or Western method
Determine the expression of the protein.
5. purposes according to claim 4, wherein when determining the expression of protein using mass spectrometry method, including from
Samples subjects, protein isolate matter preferably in urine, it is also preferable to include the protein in sample digestion, preferably use trypsase
The step of protein in sample digestion.
6. purposes according to claim 2, wherein the Mass Spectrometric Identification reagent is being targeted used in analysis.
7. one kind is diagnosed for subject's gestational diabetes mellitus, preferably early pregnancy diabetes, i.e. 6-10 weeks glycosuria gravidarum of gestation
The kit or chip of the diagnosis of disease, it includes indentifying substance, the indentifying substance is selected from the following any one or more:
1) it is used for the indentifying substance of nov protein homologue；
2) it is used for the indentifying substance of peroxiredoxin -5；
3) it is used for the indentifying substance of hepatocyte growth factor-like protein；
It is preferred that wherein the indentifying substance is Mass Spectrometric Identification reagent, preferably the Mass Spectrometric Identification reagent, antibody of claim 1 or
Its antigen-binding fragment, wherein the antibody is preferably monoclonal antibody, the antigen-binding fragment is preferably included, but is not limited to:
Fab, Fab', (Fab') 2, Fv, ScFv, bispecific antibody, three specific antibodies, four specific antibodies, double-scFv.
8. kit according to claim 7 or chip, wherein the Mass Spectrometric Identification reagent is used in targeting analysis
, it is preferable that wherein the indentifying substance is used for the urine of the subject.
9. the kit or chip of claim 7 or 8 are preparing the gestational diabetes mellitus for diagnosing subject, preferably early pregnancy
Diabetes, i.e. purposes in the composition of 6-10 weeks gestational diabetes mellitus of gestation,
It is preferred that the composition is for detecting any one of selected from the following or multinomial protein in the wherein sample of the subject
Expression；
It is preferred that the following any one of above-mentioned one or more protein expression levels or the multinomial instruction subject are with gestation
Diabetes:
1) compared to normal healthy controls, the expression of nov protein homologue is increased；
2) compared to normal healthy controls, hepatocyte growth factor-like protein expression is increased；
3) compared to normal healthy controls, the expression of peroxiredoxin -5 is reduced.
10. diagnosing the gestational diabetes mellitus of subject, preferably early pregnancy diabetes, i.e. the side of 6-10 weeks gestational diabetes mellitus of gestation
Method the described method comprises the following steps:
A. sample is obtained from the subject, it is preferable that sample is urine,
B. optionally, protein included in it is separated from sample,
C. it is determined using the kit or chip of the indentifying substance of claim 1 or claim 11-16 and comes from the subject
Sample in any one or more protein selected from the following expression: nov protein homologue, peroxiredoxin-
5, hepatocyte growth factor-like protein,
D. the expression and normal healthy controls are compared into the glycosuria gravidarum diseased state to determine subject, it is preferable that wherein
Any one of selected from the following or multinomial instruction subject suffers from gestational diabetes mellitus in sample from the subject:
1) compared to normal healthy controls, the expression of nov protein homologue is increased；
2) compared to normal healthy controls, hepatocyte growth factor-like protein expression is increased；
3) compared to normal healthy controls, the expression of peroxiredoxin -5 is reduced.
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| CN112881689A (en) * | 2019-11-29 | 2021-06-01 | 张曼 | Application of urine blood coagulation factor IX protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes |
| CN112904014A (en) * | 2019-12-04 | 2021-06-04 | 张曼 | Application of urine TBC1D5a protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes |
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| CN112924681A (en) * | 2019-12-05 | 2021-06-08 | 张曼 | Application of urine KRT10 protein and polypeptide fragment thereof in normal pregnancy |
| CN113514634A (en) * | 2020-04-09 | 2021-10-19 | 武汉市朗典精医生物科技有限公司 | Preparation of double-index immunochromatography kit for detecting Adipo/SHBG |
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| CN112924682B (en) * | 2019-12-05 | 2023-02-10 | 张曼 | Application of urine fibrinogen alpha chain protein and polypeptide fragment thereof in normal pregnancy |
| CN113514634A (en) * | 2020-04-09 | 2021-10-19 | 武汉市朗典精医生物科技有限公司 | Preparation of double-index immunochromatography kit for detecting Adipo/SHBG |
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