CN1304843C - Protein fingerprint method for biological sample analysis - Google Patents
Protein fingerprint method for biological sample analysis Download PDFInfo
- Publication number
- CN1304843C CN1304843C CNB031483755A CN03148375A CN1304843C CN 1304843 C CN1304843 C CN 1304843C CN B031483755 A CNB031483755 A CN B031483755A CN 03148375 A CN03148375 A CN 03148375A CN 1304843 C CN1304843 C CN 1304843C
- Authority
- CN
- China
- Prior art keywords
- protein
- sample
- analyte
- adsorbent
- chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to a protein fingerprint method for biological sample analysis and a protein fingerprint method for trapping proteomes for analysis by a retention chromatographic method. In the methods, proteomes are identified and detected by a mass spectrometry. The mode of analyzed data is a bar code format (a protein fingerprint) which can be read by a computer. The methods of the present invention can be used for the diagnosis of cells and clinical diseases, such as tumor diagnosis. The methods have the advantages of accuracy, convenience and rapidness.
Description
Technical field
The present invention relates to protein analysis method in a kind of new biological sample, specifically refer to be used for the protein fingerprint method of biological sample analysis.The invention still further relates to the protein fingerprint method of being detained chromatography capture protein group and adopting the barcode standard (protein fingerprint) of embodied on computer readable to analyze.
Background technology
No matter the normal function or the pathology characteristic that are cell all are somewhat dependent upon the expressed protein function of cell.Therefore, the difference of expressed protein can be used for diagnosing the illness, and finally is used for drug development and disease treatment in the identification of cell.And to carry out the differentiation analysis of protein expression and function, requirement can reach the degree of differentiating the complex mixture of molecule in the cell.But many materials often exist with trace in the cell, and the method that is used for analyzing proteins at present has limitation at above-mentioned everyway, is difficult to carry out qualitative evaluation and quantitative test with these conventional meanses.
As, a kind of molecular separation process commonly used is gel electrophoresis.Normally use isoelectric focusing elder generation isolated protein in the gel, use SDS-PAGE (SDS-PAGE) to carry out the second time again and separate.The result obtains a figure who distinguishes protein according to isoelectric point scope (net charge) and size (quality).Though useful, there is the limitation of following several respects in this method.At first, this method only provides two feature one quality and the isoelectric point (pI) of biomolecule.Secondly, the resolution of each dimension (DIMENSION) is subjected to the restriction of gel resolution characteristic.For example, usually be difficult to distinguish mass discrepancy less than 5% or the biomolecule of pI difference.The 3rd, the capacity of gel and sensitivity are all limited, possibly can't detect the biomolecule of a small amount of expression.The 4th, can't observe small protein or little peptide that molecular weight is lower than about 10-20kDa.
And for example, the clinical diagnosis disease conventional method is the known mark that requirement detects disease specifically.But preparation specificity incorporation of markings and the reagent that can identify mark in the potpourri of complexity need the plenty of time, and this has hindered the development of this type of diagnostic method.
Other analytical approach may overcome one of above limitation or several, but is difficult to they effective combinations.For example, analyze chromatography and can interact separation of biomolecules, analyze difficult and time-consuming but make various dimensions (MULTI-DIMENSIONAL) according to multiple analytes/adsorbent.And the sensitivity of this method is also very limited.
Measuring protein with mass spectral method is newer method, but present mass spectrometric analysis method can only carry out qualitative analysis to protein, can't carry out quantitative test, especially is being potpourri complexity in the sample, under the minimum situation of protein content.
Being detained chromatography is a kind of new bioanalytical method (Singh TK, Fox PF, HealyA.J Dairy Res.62, the 629-640 of the exploitation nineties in last century; 1995 and Siegel MM, Tabei K, Bebernitz GA, Baum EZ.J Mass Spectrom.33,264-273; 1998), the someone uses it in the research of molecular biology and cell biology.Earlier testing sample is combined with the adsorbent of selecting in the delay chromatography, detect the analyte that is trapped on the adsorbent then.Different with conventional chromatography, need not when being detained chromatography the first wash-out from the adsorbent of analyte, but directly detect.Its advantage is to tell analyte from the complicated sample potpourri exactly.But do not see the useful delay chromatography of document at present and carry out the protein fingerprint identification and analysis.
The protein fingerprint technology comes from potato/potato cultivar identification (Desborough S and Peloquin SJ, American Potato Journal.45,220-229 the earliest; 1968 and Desborough S and Peloquin SJ, Theoretical and Applied Genetics.39,43-47; 1969).Distinguished branch different cultivars potato with electrophoretic techniques at that time.Because the potato of different cultivars has different protein fingerprint combinations under electrophoresis.Because human body cell albumen is more than the potato complexity, so only come the protein fingerprint of identifying disease can not meet clinical needs far away with electrophoresis.
Summary of the invention:
One of purpose of the present invention is the protein group that accurately identifies in the biological sample.Two of purpose of the present invention is to be based upon the method that captures required testing protein in the complex mixture of molecule in the cell.Three of purpose of the present invention is to set up a kind of new method that detects disease in biological sample.
The present invention adopts the protein fingerprint method that protein group is differentiated.
The protein fingerprint method is by differentiating that detecting the particular proteins group is used for medical diagnosis on disease.The definition of proteomics (Proteomics) is that research one histone matter is at the lifelong change of Expression state of cell.One of purpose of proteomics is to identify and description because the organic-biological molecule of different cell differential expressions.By expression relatively can identification of cell in the difference of expressed protein.Identify the protein group that can be used as certain stigmata, can be used for the diagnosis of this disease and the screening of medicine.
The method that discriminating of the present invention detects protein group is a mass spectroscopy.
The present invention tells and captures the protein group that is used to detect exactly with being detained chromatography from the complicated sample potpourri.Being detained chromatography is that sample is contacted with the multiple adsorbent that is used for the desorption spectrum analysis/eluant, eluent combination, utilize other to separate thus and there is the different analyte of situation (that is the protein of differential expression in two parts of cell or tissue liquid extracts) in the detection system high information resolution characteristic evaluation that is beyond one's reach in two samples.Chemical characteristic according to adsorbent/eluant, eluent combination is measured the protein of the physicochemical characteristics that comprises molecular weight.Detailed description this method is as follows:
The I adsorbent
Comprise negative ion, kation, hydrophilic, hydrophobic or coordination covalency metal-chelator effect adsorbent, these methods in the separating bio chemistry and have the purposes (be that anion adsorbent is caught cationic protein, be detained on the coordination covalency metal-chelator in the explanation polypeptide analysis thing and have histidine residues) of diagnosis reverse side by the adsorbent that these methods produce.In particular, can develop the agent of chemical-biological specific adsorption to detect the important diagnostic mark.In certain embodiment, matrix can have and is to diagnose the illness or adsorption site that the used marker combination of syndrome is selected for use is arranged.
The II information processing
Detecting the analyte be adsorbed combination under the particular elutriated condition provides the information of analyte in the relevant sample and chemical feature thereof.Suction-operated is somewhat dependent upon the binding characteristic of adsorbent.The analyte that combines with adsorbent has the feasible characteristic that is combined into possibility.For example, be that cationic molecule has under the elution requirement of this pH and will combine with anion adsorbent under the specific pH.The strong cation molecule is only at wash-out from the adsorbent just under the very strong elution requirement.So, in the sample specific analyte under the particular elutriated condition in conjunction with certain adsorbent determine not only by the separated from one another of analyte with differentiated the analyte in the potpourri with the separating of analysis that does not have in conjunction with required corresponding chemical characteristic, and identified a class or single analyte with particular chemical characteristic.Gather relevant analyte and not only can differentiate analyte in the potpourri in detail in the delay information on one or more adsorbents under the multiple elution requirement, and the chemical information of relevant analyte is provided, these information itself just can be finished the evaluation to them.This data are called as " delay data ".
The data that draw with programmable calculator analysis delay test are the simplest.Computer program generally comprises a computer-readable recording medium that stores coding.Certain computer code enters storage, has wherein comprised various positions of levying a little on the matrix array, the absorption at this place and be used for washing the elution requirement of adsorbent.So identifying, available these information of program determine certain optionally stack features on the array.Computing machine also comprises such coding, but they accept the signal strength data from the different molecular weight of specific addressable location on the probe.The quantity of these data indication institute cls analysis things may comprise the signal intensity of each analyte of surveying and the molecular weight that records.
Computing machine also has the coding of deal with data.Among one of embodiment, disposal route relates to analyte recognition feature overview of formation.For example, can be with the data based specific binding characteristic of delay (for example with the anion adsorbent) classification of the specific analyte that records according to molecular weight.The data of collecting provide the overview of certain specific analyte chemical feature.Retention characteristics reflection analyte function, the latter is reflect structure then.For example, can draw the isoelectric point of certain protein according to the delay horizontal data institute information revealed on multiple kation and anion adsorbent under the different pH elution requirements.This so reflect the roughly quantity of ion amino acid in this protein.So computing machine may comprise the coding with combining information transferring structure information.And to secondary treating (for example posttranslational modification) the change recognition feature of analyte, this can be reflected by combination or mass discrepancy.
The data that draw are protein fingerprint, and it can show by various forms.In a kind of form, signal intensity is shown as the functional arrangement of molecular weight.Another kind of form claims " barcode standard " again, and wherein, signal intensity shows with the darkness intensity level along linear axes, draws outer appearnce and reads trade name like used bar code band on the market.
III is detained the combination that chromatography relates to multiple separation method, comprises parallel detection and describes multiple analytes.These combined methods serve many purposes.Development target analyte detection method; Differential protein is showed; The toxicology screening; Detect in the time of multiple diagnostic flag; Drug development.
Embodiment is described in detail this method.Cell sample comprises the protein of counting in 100,000 usually.People may wish accurately to tell each target protein in the sample.The first step is set up the delay figure of target analyte with the multiple choices condition.For example, adsorbent can be an anionite.
Figure can pick out the alternative condition that at least a target analyte is retained from this delay.Can select the selective conditions of target analyte generation maximum combined.But,, then should select target analyte absorption and off-peak condition if this selective conditions is stronger to other selective conditions of selectivity ratios of target analyte.For example, suppose to be detained map analysis and show that near neutral pH, target analyte is kept by anion exchange, but is adsorbed in the water wettability adsorbent also.
The method of the analyte of IV identification of organism storeroom differential expression
Another aspect of the present invention content provides the organic-biological molecule, particularly method of protein of identifying differential expression in two or more samples." differential expression " refers to the difference on two sample room amount of analyte or the matter.These differences may be because of arbitrary stage of protein expression, and this method has been utilized the extraordinary resolution characteristic and the sensitivity of being detained chromatography.At first, use the resolution of same group selection condition preparation high more, so comparable analyte quantity is also many more.Then, relatively discern figure to identify the analyte that is kept by two groups of adsorbent differences.Difference is detained and is comprised quantitative delay.Just regulation and control or negative regulation that this prompting is expressed.Difference is detained the difference that also comprises amalyzing substances.For example, the difference of protein post-translational modification can cause the difference of identification figure, shows as binding characteristic difference (for example, if protein by glycosylation and agglutinin in conjunction with different) or mass discrepancy (for example difference of translation back cutting) during detection.This analysis also can be carried out with programmable calculator.
This method is specially adapted to detect the albumen of two kinds of cell differential expressions.Two kinds of cells can be for example cancer cells of normal cell and pathological cells, or the cell of varying level, or different the growth or the cell of differential period, or the cell of different piece in the cell cycle.But.This method also can be used to detect the allogenic cell under the different condition.In such method, a biological sample contacts with test agent, and another part do not contact.Then, the delay figure that compares both.According to the difference of retention characteristics or quality, this method can show the increase or the minimizing of protein or other biological developed by molecule, or certain change has taken place.
Utilize and be detained the differential expression protein physicochemical property information that figure obtains, can measure the candidate feature of protein with the inventive method.
This method is applicable to the diagnostic flag of identifying disease.Compare with normal specimens or cell, the protein of differential expression may be exactly diagnostic flag in patient's sample or the pathology cultured cell.
V improves sensitivity by the kalabolism amplification of signal
Large protein is split into small fragment to be detected these small fragments then and can significantly improve and detect there be (for example, because differential expression) in large protein in complex mixture difference sensitivity.The raising of sensitivity has several reasons.At first, when all proteins in the sample all by (for example) enzymolysis and during fragmentation, large protein may produce more multi-disc section rather than small protein.Secondly, when the overall sensitivity of desorption spectrum method is higher than high molecular when low-molecular-weight.The 3rd, protein fragmentsization has increased the number of signals from target analyte, has therefore improved the possibility that records analyte.The 4th, protein fragmentsization has improved catches possibility, has therefore improved the possibility that records target analyte.The 5th, if protein difference in two kinds of samples exists, by increasing the number of signals from this albumen, the difference of amount more may be recorded so.
Disease protein mark in the human body tends to be subjected to the degraded of enzyme in the blood.Like this, we just can catch this kalabolism signal with the delay chromatography, thereby improve the sensitivity of protein fingerprint.The instantiation that is detained the analyte that chromatography differentiates comprises biomacromolecule, peptide for example, protein, enzyme, sugar, oligosaccharides, polysaccharide; The fragment of above-mentioned biomacromolecule, for example fragments of peptides and protein fragments; Above-mentioned biomacromolecule compound, protein-DNA compound, receptor-ligand compound, enzyme-substrate, enzyme inhibitor, peptide complexes, protein complex, saccharide complex and polysaccharide compound; Biological micromolecule, amino acid for example, medicine, hormone, synthesized micromolecule, for example active drug in pharmacy or the treatment.
The invention provides a kind of unified operating system, be used for protein function, the discovery and the diagnosis of cell function and biological integral function.
Ability (for example negative ion/kation, hydrophobicity/water wettability, or the effect of specific biological molecular recognition) separate analytes at least two kinds of first different dimensions of under at least two kinds of different selection conditions, adsorbing in particular, stationary phase according to analyte.Then, use desorption spectrum (for example laser desorption mass spectrum), the analyte that further detection has separated according to quality separate analytes on second dimension.The adsorbed character of analyte provides the physical chemistry information of analyte.
In an embodiment, described method comprises: I) analyte contacts with first selective conditions (adsorbent) on the accurate location, makes analyte be adsorbed agent and keeps.II) under first selective conditions, detect the analyte that is detained with desorption spectrum.III) detect step and comprise the quality of using laser desorption mass spectroscopy analyte.
The difference of elution requirement is pH, surge capability, ionic strength, water-bound feature, scale remover kind, scale remover intensity, hydrophobicity or specific inductive capacity.
The invention provides a kind of determine certain analyte in first and second product whether difference have the method for (for example differential expression).This method can be used for showing the combined method that detects the differential protein expression by differential protein.This method comprises A) the determination and analysis thing delay of first under at least a selective conditions figure in first sample; B) determination and analysis thing second under identical selective conditions in second sample is detained figure; And C) compares first and second differences of being detained between the figure.The difference of two delay figure has affirmed that the difference of analyte in first and second samples exists.
Whether one of embodiment measures certain albumen at two kinds of different samples, comprises first and second samples of cell or from differential expression in the material of cell.In an embodiment, first biological sample is from healthy person, and second biological sample is from the patient of certain illness.Sample can be selected from for example blood, urine, serum and tissue.With the analyte that in patient's sample, increases as the candidate diagnostic flag.Usually, confirm that a kind of diagnostic flag needs to detect this label in a plurality of experimenters.
The present invention on analyzable sample type without limits.The present invention is specially adapted to differentiate biological sample, especially the analyte in biofluid and the extract; Also be specially adapted to differentiate analyte, especially organic molecule in the abiotic organic composite and the composition of inorganic molecule.
The concrete operations step of being detained chromatography and protein fingerprint method:
It below is an operation scheme example with delay chromatography provided by the invention and protein fingerprint method.
1. sample preparation
Biological sample is diluted in the solution.Centrifugal clarification sample optionally.
2. go up sample
With the sample point sample on site of matrix (comprising negative ion, kation, hydrophilic, hydrophobic or coordination covalency metal-chelator).
3. washing
With in conjunction with solvent wash.Before the sample bone dry, first part of 2uL wash solution is added to this site.Wash solution stopped on the site 15 seconds at least.With transfer pipet inspiration 10 times.Thoroughly remove first part of wash solution, repeat above step with second part of 2uL cleansing solution.
Water is the whole array of washing thoroughly.
The air dry chip.
Add 0.5uL energy-absorbing molecule SINAPINIC acid (with 50% acetonitrile, the standard solution of the preparation of 0.5% trifluoroacetic acid).
The air dry chip.
Analyze the protein in array analysis delay and each site with nitrogen laser (335nm) and 80cm tof tube with laser desorption/ionization time of-flight mass spectrometer.Carry out the overlapping displaying of data with the Computer Analysis data.
The present invention is detained the diagnosis that chromatography and protein fingerprint method can be used for cell and clinical disease, as the diagnosis of tumour.
China has the carrier of 1.3 hundred million hepatitis B at present, the cirrhosis that causes comprising 2,000 3 hundred ten thousand hepatitis B.Hepatocellular carcinoma is mainly caused by the cirrhosis that hepatitis B causes.The specificity and the susceptibility of alpha-fetoprotein kit that is used for auxiliary diagnosis liver cancer at present is all very poor, can't be used to differentiate liver cancer and cirrhosis.Can differentiate normal cirrhosis and the liver cancer that causes by hepatitis B that reaches with this method, see embodiment for details.
The characteristics of method provided by the invention are:
(1) accurate
Characteristics of being detained chromatography are to tell analyte from the complicated sample potpourri exactly.What detect in the delay chromatography is the analyte that is trapped on the adsorbent.So being detained chromatography can provide the relevant direct information that is detained analyte chemical or architectural feature.
Be detained chromatography and conventional chromatography and have following difference.What at first, detect in the delay chromatography is the analyte that is trapped on the adsorbent.In conventional chromatography, analyte is wash-out from the adsorbent earlier, detects then.Can not detect the analyte under the wash-out on the adsorbent with conventional method.The second, adsorption chromatography detects to combine with maldi mass spectrometer provides the good sensitivity of femto mole level and high resolution.The 3rd, to a certain extent, because allow direct check and analysis thing, the delay chromatography provides the ability with multiple different choice condition express analysis retentate, and the quick dimension description of analyte in various product is provided thus.The 4th, adsorbent can be in predetermined arrangement, but addressable location is attached on the matrix.This just can carry out parallel processing to the analyte of the different absorption positions of contact (that is, " affinity position " or " site ") under different elution requirements.
Mass spectrum is directly analyzed very strong accuracy, and general error rate has only 0.1Da.For example, detect the 3935Da that has positive electricity and the albumen of 8939Da and caught by the matrix of anionic surface, because the mass spectrum rate has only 0.1Da, there are second kind of protein in positive electricity protein 39 35Da and 8939Da.Because protein by amino acid form and amino acid whose average quality is 100Da, therefore, the above gap of 50Da must be arranged, just may be second kind of protein.
(2) convenient
This method has been divided into several big classes with haemocyanin, i.e. negative ion, kation, hydrophilic, hydrophobic or coordination covalency metal-chelator action protein.Like this, available mass spectrometer is directly analyzed.
(3) quick
When carrying out the disease blood serum diagnosis, need not protein is checked order with protein fingerprint method provided by the invention.The present invention has adopted computing machine " barcode standard ", and signal intensity shows with the darkness intensity level along linear axes.This intensity can be analyzed the contrast power automatically with the scanner record and with analysis software, thereby help the diagnosis of clinical complexity, carry out analysis of variance, the mutation analysis of tulase persister in the crowd of forensic analysis, crowd's gene etc. as available this " protein fingerprint " or barcode standard.
Embodiment
Cirrhosis that embodiment 1 hepatitis B causes and liver cancer are distinguished
(1) experimental technique
1uL serum is added 9M urea, under 4 ℃-6 ℃, make protein and urea effect 30 minutes.Then above-mentioned sample is added in conjunction with solvent (50mM sodium acetate, pH 4.0～6.0) and be called for short among the BB. then, rare serum is added the anion adsorbent (anion adsorbent on the steel matrix of monox bag quilt: SO
3 -, i.e. anion exchange adsorbent) chip on, at room temperature be with anionic chips incorporate.Wash twice with BB, wash twice with other dH2O of HPLC level then.Allow the chip air dry.SINAPINIC acid (5mg/mL 50% acetonitrile that adds 0.5uL; 0.5% trifluoroacetic acid), the drying of giving free rein to.Directly analyze with mass spectrometer.Result's Computer Analysis mass spectrometric data.
(2) experimental result
Use statistical method, the form of analyzing the gained data is that protein fingerprint is shown as the darkness intensity level signal along linear axes with the machine-readable barcode standard of getting of calculating, and this intensity can be analyzed the contrast power automatically with the scanner record and with analysis software.By analyzing several thousand kinds of haemocyanin fingerprint peakses, find that following five kinds of protein fingerprints can be used to distinguish cirrhosis and liver cancer (P is less than 0.01).
2045Da
3935Da
4469Da
8687Da
8933Da
With five known protein fingerprints, double-blind 40 routine hepatitis B cause the liver cancer test that cirrhosis and hepatitis B cause.
| Liver cancer (30) | Cirrhosis (40) | |
| Liver cancer | 27 | 6 |
| Cirrhosis | 3 | 34 |
Susceptibility 90% specificity 85%
(3) conclusion
To compare from sample and the normal specimens of the patient group with statistical significance, five kinds of haemocyanin fingerprints with the negative ion chip is caught can be used for antidiastole.The methods of treatment of cirrhosis is an expectant treatment, and liver cancer must be passed through operative treatment, and this method accuracy rate is 85-90%.Therefore, this is present best non-invasive diagnostic method.
Embodiment 2 normal and liver cancer differentiations
(1) experimental technique
1uL serum is added 9M urea, under 4 ℃-6 ℃, make protein and urea effect 30 minutes.Then above-mentioned sample is added in conjunction with solvent (50mM sodium acetate, pH 4.0～6.0) and be called for short among the BB.Then, rare serum is added the anion adsorbent (anion adsorbent on the steel matrix of monox bag quilt: SO
3 -, i.e. anion exchange adsorbent) chip on, at room temperature be with anionic chips incorporate.Wash twice with BB, wash twice with other dH2O of HPLC level then.Allow the chip air dry.SINAPINIC acid (5mg/mL 50% acetonitrile that adds 0.5uL; 0.5% trifluoroacetic acid), the drying of giving free rein to.Directly analyze with mass spectrometer.Result's Computer Analysis mass spectrometric data.
Use statistical method, the form of analyzing the gained data is that protein fingerprint is shown as the darkness intensity level signal along linear axes with the machine-readable barcode standard of getting of calculating, and this intensity can be analyzed the contrast power automatically with the scanner record and with analysis software.By analyzing several thousand kinds of haemocyanin fingerprint peakses, find that following three kinds of protein fingerprints can be used for distinguishing normal and liver cancer (P is less than 0.001).
3158Da
4793Da
5812Da
With three known protein fingerprints, the normal and liver cancer test of double-blind 30 examples.
| Liver cancer (30) | Normally (30) | |
| Liver cancer | 27 | 2 |
| Normally | 3 | 28 |
Susceptibility 90% specificity 93.3%
Embodiment 3 cirrhosis normal and that hepatitis B causes are distinguished
(1) experimental technique
1uL serum is added 9M urea, under 4 ℃-6 ℃, make protein and urea effect 30 minutes.Then above-mentioned sample is added in conjunction with solvent (50mM sodium acetate, pH 4.0～6.0) and be called for short among the BB. then, the serum that has diluted is added the anion adsorbent (anion adsorbent on the steel matrix of monox bag quilt: SO
3 -, i.e. anion exchange adsorbent) chip on, at room temperature be with anionic chips incorporate.Wash twice with BB, wash twice with other dH2O of HPLC level then.Allow the chip air dry.SINAPINIC acid (5mg/mL 50% acetonitrile that adds 0.5uL; 0.5%TFA), the drying of giving free rein to.Directly analyze with mass spectrometer.Result's Computer Analysis mass spectrometric data.
Use statistical method, the form of analyzing the gained data is that protein fingerprint is shown as the darkness intensity level signal along linear axes with the machine-readable barcode standard of getting of calculating, and this intensity can be analyzed the contrast power automatically with the scanner record and with analysis software.By analyzing several thousand kinds of haemocyanin fingerprint peakses, find that following three kinds of protein fingerprints can be used to distinguish cirrhosis (P is less than 0.01) normal and that hepatitis B causes.
3158Da
3934Da
4793Da
With three known protein fingerprints, the normal cirrhosis that causes with 40 routine hepatitis B of double-blind 30 examples.
| Cirrhosis (40) | Normally (30) | |
| Cirrhosis | 35 | 3 |
| Normally | 5 | 27 |
Susceptibility 87.5% specificity 90%
Claims (4)
1. protein analysis method in the biological sample is characterized in that adopting and is detained chromatography and catches in the sample protein group and analyze, and step is as follows:
1) sample preparation: biological sample is diluted in the solution;
2) go up sample: the sample point sample is being detained on the chip on the site of chromatography substrate;
3) use in conjunction with solvent wash: before the sample bone dry first part of wash solution is added to this site, wash solution stopped on the site 15 seconds at least; With the transfer pipet inspiration several times, thoroughly remove first part of wash solution; Repeat above step with second part of cleansing solution; Water is the whole array of washing thoroughly; Air dry matrix and chip;
4) add the energy-absorbing molecular criteria solution of 0.5 μ L with 50% acetonitrile and the preparation of 0.5% trifluoroacetic acid, air dry matrix and chip;
5) with laser desorption/ionization time of-flight mass spectrometer, analyze array with nitrogen laser and tof tube, analyze the protein that is stranded in each site; Carry out the overlapping displaying of data with the Computer Analysis data.
2. the described protein analysis method of claim 1, described delay chromatography substrate comprises following adsorbent: negative ion, kation, hydrophilic, hydrophobic or coordination covalency metal-chelator.
3. the described protein analysis method of claim 1, the form of analyzing the gained data is the barcode standard with embodied on computer readable, protein fingerprint is shown as the darkness intensity level signal along linear axes, and this intensity can be analyzed the contrast power automatically with the scanner record and with analysis software.
4. the described protein analysis method of claim 1, the application programming computing machine is analyzed and is handled the gained data.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031483755A CN1304843C (en) | 2003-07-02 | 2003-07-02 | Protein fingerprint method for biological sample analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031483755A CN1304843C (en) | 2003-07-02 | 2003-07-02 | Protein fingerprint method for biological sample analysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1566953A CN1566953A (en) | 2005-01-19 |
| CN1304843C true CN1304843C (en) | 2007-03-14 |
Family
ID=34472269
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031483755A Expired - Lifetime CN1304843C (en) | 2003-07-02 | 2003-07-02 | Protein fingerprint method for biological sample analysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1304843C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101685080B (en) * | 2008-06-12 | 2012-10-31 | 高尚先 | Method for analyzing proteome in biological sample |
| CN103454428A (en) * | 2012-06-05 | 2013-12-18 | 许洋 | Novel immunomic mass spectrometry kit for detecting individualized insulin and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1288955A (en) * | 1999-09-20 | 2001-03-28 | 中国科学院力学研究所 | Multielement protein chip and its preparation and application |
| WO2002056026A1 (en) * | 2001-01-09 | 2002-07-18 | Mitsubishi Pharma Corporation | Novel proteome analysis method and devices therefor |
| CN1381723A (en) * | 2002-05-16 | 2002-11-27 | 复旦大学 | Protein chip and its preparing process |
| WO2003031031A1 (en) * | 2000-11-16 | 2003-04-17 | Ciphergen Biosystems, Inc. | Method for analyzing mass spectra |
| CN1413261A (en) * | 1999-12-20 | 2003-04-23 | 宾夕法尼亚州研究基金会 | Deposited thin films and their use in detection, attachment and bio-medical application |
-
2003
- 2003-07-02 CN CNB031483755A patent/CN1304843C/en not_active Expired - Lifetime
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1288955A (en) * | 1999-09-20 | 2001-03-28 | 中国科学院力学研究所 | Multielement protein chip and its preparation and application |
| CN1413261A (en) * | 1999-12-20 | 2003-04-23 | 宾夕法尼亚州研究基金会 | Deposited thin films and their use in detection, attachment and bio-medical application |
| WO2003031031A1 (en) * | 2000-11-16 | 2003-04-17 | Ciphergen Biosystems, Inc. | Method for analyzing mass spectra |
| WO2002056026A1 (en) * | 2001-01-09 | 2002-07-18 | Mitsubishi Pharma Corporation | Novel proteome analysis method and devices therefor |
| CN1381723A (en) * | 2002-05-16 | 2002-11-27 | 复旦大学 | Protein chip and its preparing process |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1566953A (en) | 2005-01-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105823877B (en) | Biomarkers for ovarian cancer: CTAP 3-related protein | |
| JP6010597B2 (en) | Biomarkers for ovarian cancer | |
| Yu et al. | Prediction of pancreatic cancer by serum biomarkers using surface-enhanced laser desorption/ionization-based decision tree classification | |
| US8389222B2 (en) | Apolipoprotein fingerprinting technique and methods related thereto | |
| CN1649613A (en) | Serum biomarkers in hepatocellular carcinoma | |
| CN101002084A (en) | Biomarkers for peripheral artery disease | |
| Gulcicek et al. | Proteomics and the analysis of proteomic data: an overview of current protein‐profiling technologies | |
| CN101093215A (en) | Mass spectrum kit and method for evaluating prognosis from screening lung cancer | |
| Helena et al. | Current applications of capillary electrophoresis‐mass spectrometry for the analysis of biologically important analytes in urine (2017 to mid‐2021): A review | |
| CN1304843C (en) | Protein fingerprint method for biological sample analysis | |
| Wu et al. | Proteomic evaluation of urine from renal cell carcinoma using SELDI-TOF-MS and tree analysis pattern | |
| US20120046185A1 (en) | Panel of biomarkers for ovarian cancer | |
| CN115452521A (en) | Marker, product and system for identifying echinococcosis hepatica | |
| CN1936578A (en) | Use of protein finger print method in detecting kidney cancer cell protein finger print in blood | |
| CN1595139A (en) | Use of protein fingerprint method in detecting protein fingerprint of gastric carcinoma cell in blood | |
| CN1595138A (en) | Use of protein fingerprint method in detecting protein fingerprint of colon cancer and rectum cancer in blood | |
| CN111748624B (en) | Biomarker for predicting whether liver cancer is recurrent | |
| Zhong et al. | Proteomic profiling of ovine serum by SELDI-TOF MS: optimisation, reproducibility and feasibility of biomarker discovery using routinely collected samples | |
| CN1310033C (en) | New method for detecting esophageal cancer haemocyanin fingerprint | |
| CN101021508A (en) | Application of end-grain cutting protein peptide A to stomach cancercell lymph node metastasis detection | |
| CN1595140A (en) | Use of protein fingerprint method in detecting protein fingerprint of pancreas cancer cell in blood | |
| CN101169416A (en) | Use of magnetic bead supported matrix and MS for judging mass spectrometry polypeptide spectrum and protein fingerprint | |
| CN1657929A (en) | Use of detecting urine protein fingerprint by protein fingerprint technique | |
| CN1844914A (en) | Application of high expression or low expression of modified protein to tumor cell detection | |
| CN1844915A (en) | Use of enhanced protein fingerprint spectrum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20070314 |
|
| CX01 | Expiry of patent term |