CN101329348A - Optimizing mass spectrogram model for detecting stomach cancer characteristic protein and preparation method and application thereof - Google Patents
Optimizing mass spectrogram model for detecting stomach cancer characteristic protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an optimum mass spectrometry model and a preparation method thereof for detecting the feature protein of gastric cancer, belonging to the field of mass spectrometry detection technique. The invention is characterized in that seven up-regulated proteins and three lower-regulated proteins are screened from the blood serum to be used as the feature proteins; any two or more proteins of the ten proteins are chosen so as to establish a blood serum feature protein mass spectrometry model of identification with two in a group for patients with gastric cancer and normal people, and patients with benign gastric cancer disease, lymphatic metastasis of gastric cancer and remote metastasis of gastric cancer according to the mass-charge ratio m/z of each protein peak and the critical peak average value of the protein; the preparation method of the invention provides a foundation for discovering new gastric cancer biological marks. The method of the invention is better than any single detection method adopted currently for the detection of the gastric cancer, provides a non-invasive technique for the early detection and early treatment of the gastric cancer, thus providing a new method for reducing the mortality of the gastric cancer, improving the cure rate of the gastric cancer and screening and examining the gastric cancer for high-risk population further.
Description
Technical field
The invention belongs to the mass spectrum detection field, particularly the Mass Spectrometer Method method that cancer of the stomach blood is optimized.A kind of by removing to catch biological marker with the magnetic bead matrix of protein bound, and detect the cancer of the stomach biological marker with the mass spectrophotometry that quantitative control is arranged.The present invention relates to the protein detection field referred in this, is a kind of external Mass Spectrometer Method method of new Noninvasive.The present invention can be applied to the detection method or the kit of the cancer of the stomach biological marker combination in the body fluid that has broken away from human body.
Background technology
Cancer of the stomach be the process that a polygenic mutation causes the activation of tumor suppressor gene inactivation, oncogene.The generation of tumour, development are very complicated and very long processes, follow the molecular changes of polygenes and albumen.Genomics research is because the restriction of self is difficult to illustrate fully gene and protein participation process and concrete effect thereof.Proteomics high-sensitivity analysis technology helps to catch those molecules trickle in the tumour evolution process and changes, and makes it to become the important clue of diagnosis and the differentiation of monitoring tumour.Clinically the malignant tumour prognosis evaluation is mainly depended on clinical manifestation, pathology and traditional tumour sign (tumor marker) at present, but the early stage recurrence of tumour or usually do not have obvious clinical manifestation when shifting, and the pathology evidence is difficult to obtain and poor repeatability.China's incidence gastric cancer rate height, its mortality ratio account for position first of the various malignant tumours again, and therefore, cancer of the stomach is the common disease of serious harm China people ' s health, should draw attention.The sensitivity of present existing tumor markers single index only is 18-40%, though the result of use in conjunction can reach 60-80%, false positive rate is higher, is difficult to as early detection.The high mortality of cancer of the stomach makes people constantly seek a kind of effective early detection method, only to detect index with a kind of albumen as serology clinically be not enough far away on susceptibility and specificity, in case imageology detects and finds space occupying lesion then be not phase very early.Disease is found extremely early stage just can the getting of taking place, and controlled and treatment, and then improve cure rate greatly or obviously improve patient's prognosis and quality of life.Along with the carrying out of clinical proteomics, make the early detection of cancer of the stomach become possibility in recent years.
The isolation technics of protein is used isolation technics in proteomics have two purposes.The first, become single protein or protein group to simplify complicated protein mixture by mixture separation with protein; The second, the method by mark can compare the different manifestations of protein in the blend sample of two protein.Wherein major technology has dielectrophoresis (two-dimensional gel electrophoresis, 2-DE), high performance liquid chroma-tography (high-performanceliquid chromatography, HPLC), Capillary Electrophoresis (capillary electrophoresis, CE), affinity chromatography (affinity choromatography), protein chip (protein microarray), magnetic microsphere (magneticbeads is called for short magnetic bead) and immune group etc.Particularly two new protein fingerprint spectral technologies of protein chip and magnetic bead have overcome the shortcoming of conventional art, but have characteristics such as high sensitivity, high flux, good reproducibility mechanized operation as a result and method be flexible.The testing sample wide material sources need not done special pre-treatment, and directly point sample detects, as serum, urine and tissue fluid etc.; Detect fast, only need about 5 minutes the detection time of a general routine sample, from sample preparations to going out as a result overall process only about 1 hour.Two new protein fingerprint spectral technologies of protein chip and magnetic bead might potential source biomolecule sign (biomarker) context of detection be created the revolutionary (Xu Yang of breakthrough in body fluid, the progress of protein fingerprint pattern technology in laboratory diagnosis and clinical medicine, preclinical medicine and clinical, 2007,27:134-142).Ground substance assistant laser parsing/ionization time of flight mass spectrometry (matrix-assisted laser desorption/ionization time of fight massspectrometry, MALDI-TOF-MS) with protein chip isolation technics use in conjunction be surface-enhanced laser parsing/ionization time of flight mass spectrometry (surface enhanced laser desorption/ionization time of fight massspectrometry, SELDI-TOF-MS).2-DE is used to clinical protein science research at first, but its resolution to hydrophobicity, highly acid and strong alkali albumen is not enough, and can not detect low-abundance protein, and historical facts or anecdotes is limited with being worth.Recently, protein chip combines with mass-spectrometric technique (SELDI-TOF-MS), has been successfully used to tumor research; But because the bonded area of magnetic bead surfaces is far longer than protein chip, thereby the sensitivity of magnetic bead is than protein chip higher (Liu Jiandong etc., hematoglobin protein group and mass spectrometer testing process standardization pre-test.Preclinical medicine and clinical, 2007,27:193-197).Magnetic bead has the advantages that to be better than two dimensional electrophoresis, protein chip and other mass spectrometry methods, (it is good etc. to slaughter generation to be widely used in the research such as examination of knubble biological flag, the detection of carcinomebryonic antigen negative colorectal cancer and colorectal cancer prognosis associated biomolecule sign, preclinical medicine and clinical, 2007).Characteristics such as that this technology has is easy and simple to handle, need not centrifugal sample, can directly analyze primeval life sample (as serum, urine etc.), the sample consumption is little, be fit to the parallel detection of various product simultaneously and directly carry out the search and the analysis of protein panorama type, particularly small molecular weight protein and low-abundance protein had higher capture effect, can with other proteomics method complementations, be widely used at present the examination and the clinical detection of tumor-marker, and all obtained good result.But the domestic report that does not still have employing magnetic bead and mass-spectrometric technique acquisition detection cancer of the stomach serum characteristic protein so far.
Summary of the invention:
The objective of the invention is for overcoming weak point present cancer of the stomach serum detection technique, set up a kind of optimization mass spectra model that detects stomach cancer characteristic protein and its production and application, this model provides new approaches and methods for early detection, and for finding that further new knubble biological flag provides the foundation.
The mass spectra model of the detection cancer of the stomach serum proteins that the present invention proposes is characterized in that: filter out 7 upregulated proteins and 3 down-regulation proteins as characteristic protein from serum; Choose any two or more albumen in above-mentioned 10 characteristic proteins, according to the mass-to-charge ratio m/z value of each protein peak and with the threshold peak average M of this albumen, according to the mass-to-charge ratio m/z value of each protein peak and with the threshold peak average M of this albumen, set up patients with gastric cancer and normal person, stomach benign disease, lymph nodal metastasis and cancer of the stomach far-end and shifted the serum characteristic protein detection mass spectra model (Fig. 3-5) that the patient differentiates in twos, said specific protein mass-to-charge ratio m/z and threshold peak average M are respectively m/z=1465, M 〉=10.21; M/z=5089, M 〉=8.29; M/z=11471, M 〉=13.23; M/z=6443, M≤2.76; M/z=1465, M 〉=16.77; M/z=8936, M 〉=10.71; M/z=11680, M 〉=21.64; M/z=8892, M 〉=11.56; Molecular weight (m/z) error＜0.01%, CV＜5～10% of threshold peak average M.
The present invention proposes the preparation method of above-mentioned detection cancer of the stomach haemocyanin mass spectra model, may further comprise the steps:
1) 4 ℃ of conditions are collected the patients with gastric cancer serum of clarifying a diagnosis through pathology and the serum that is defined as normal health person through health check-up as two groups of serum specimens in following 2 hours, and it is standby to carry out-80 ℃ of cryogenic freezings;
2) adopt WCX magnetic bead or C8 and C18 hydrophobic matrix magnetic bead that the albumen of two groups of serum specimens of described patients with gastric cancer and normal person is adsorbed;
3) use the mastrix-assisted laser desorption ionization time of flight mass spectrometer, with nitrogen laser (337nm) and 80cm or 120cm tof tube analysis array, or with liquid chromatography mass combined instrument (LC-MS) two groups of haemocyanins that are combined on the magnetic bead being read behind the biological marker of electron spray ionisation wash-out, setting the highest detection molecular weight is 50kDa, optimize molecular weight ranges 1000～15000Da, optimal accumulated center 8000Da, data acquisition parameters scope 20～80, laser intensity 150-180, detection sensitivity is 7-8, collection adds up to 130 times, obtains two histone mass-spectrograms thus;
4) before each experimental data is collected, rectify an instrument with All-in-one standard protein and mass spectral standardization Quality Control O type serum, with 2746 ± 1Da, 5909 ± 1Da, 6634 ± 1Da base peak is demarcation property (molecular weight) quality control standard in the mass spectrum, make molecular weight error＜0.01%, thus the accurate protein spectrum collection of illustrative plates that obtains;
5) quantitative property spectrum regulation and control: before each test, with mass spectral standardization quality controlled serum (slurry), the maximal value that quantitative standards peak 6634.0Da equal strength transfers to 50% signal intensity will be used in the standardization quality controlled serum, make CV＜5～10% of threshold peak average M, and collect accurate mass-spectrogram data;
6) the gained data are carried out Mann-Whitney U test statistics and handle, (obtain 213 protein peaks altogether, assert that the P value is less than 10 according to the difference of albumen peak value between patients with gastric cancer and the normal human serum
-5The time have a statistical significance), CV＜5～10%; Initial analysis filters out patients with gastric cancer and normal population has 10 difference polypeptide proteins, and the expression variation of the molecular weight at different peaks, threshold peak average M and characteristic protein sees Table one:
The expression of table one patients with gastric cancer characteristic protein changes
The expression of molecular weight of albumen (m/z) patients with gastric cancer albumen threshold peak characteristics of mean albumen changes
M±SD
1465 10.21±6.29 ↑
5089 8.29±6.27 ↑
5916 7.31±5.93 ↑
8892 11.56±8.33 ↑
11471 13.23±12.22 ↑
11680 21.64±17.12 ↑
8936 10.71±8.31 ↑
17250 3.71±3.05 ↓
6443 2.76±2.81 ↓
8696 2.87±1.45 ↓
In the table one: to upward arrow " ↑ " is the rise characteristic protein of high expressed in patients with gastric cancer serum; Arrow " ↓ " is a low downward modulation characteristic protein of expressing in patients with gastric cancer serum downwards;
With the characteristic protein of above-mentioned 10 differential proteins as cancer of the stomach mass spectrum inspection model;
6) because a plurality of characteristic proteins combine just cancer of the stomach and normal person etc. can be separated fully, so choose any two or more albumen in above-mentioned 10 characteristic proteins, according to the mass-to-charge ratio m/z value of each protein peak and with the threshold peak average M of this albumen, set up patients with gastric cancer and normal person, stomach benign disease, lymph nodal metastasis and cancer of the stomach far-end and shifted the serum characteristic protein detection mass spectra model (Fig. 3-5) that the patient differentiates in twos, said specific protein mass-to-charge ratio m/z and threshold peak average M are respectively m/z=1465, M 〉=10.21; M/z=5089, M 〉=8.29; M/z=11471, M 〉=13.23; M/z=6443, M≤2.76; M/z=1465, M 〉=16.77; M/z=8936, M 〉=10.71; M/z=11680, M 〉=21.64; M/z=8892, M 〉=11.56; Wherein the mass spectra model A that differentiates of patients with gastric cancer and normal person is by m/z=1465, M 〉=10.21; M/z=5089, M 〉=8.29 draws and forms, and the mass spectra model B of patients with gastric cancer and the discriminating of stomach benign disease is by m/z=11471, M 〉=13.23; M/z=6443, M≤2.76 are drawn and are formed, and patients with gastric cancer and lymph nodal metastasis and cancer of the stomach far-end shift the mass spectra model C of discriminating by m/z=1465, M 〉=16.77; M/z=8936, M 〉=10.71; M/z=11680, M 〉=21.64; M/z=8892, M 〉=11.56 are drawn and are formed; Molecular weight (m/z) error＜0.01%, CV＜5～10% of threshold peak average M.Utilize this mass spectra model,, just can be used for the cancer of the stomach check as long as the mass-to-charge ratio of respective egg white matter in person under inspection's serum and the threshold peak average M and the mass spectra model of the present invention of this albumen are analyzed one by one.
Utilize this mass spectra model A-C (Fig. 3-5), the mass-to-charge ratio of respective egg white matter in person under inspection's serum and the threshold peak average M and the mass spectra model of the present invention of this albumen are analyzed one by one, through clinic trial and double-blind, its susceptibility of differentiating patients with gastric cancer and normal person is 96%, and specificity is 91%; Cancer of the stomach and stomach benign disease patient susceptibility 93%, specificity 91%, patients with gastric cancer and lymph nodal metastasis susceptibility 87%, specificity 93%; Patients with gastric cancer and cancer of the stomach far-end shift susceptibility 81%, specificity 93%.
Utilize the experimental result of C8 and C18 hydrophobic matrix magnetic bead consistent with the experimental result of above-mentioned WCX anionic substrates magnetic bead.
The present invention adopts above-mentioned mass spectra model to can be used for the assessment of cancer of the stomach early detection, examination and recurrence, transfer.
Characteristics of the present invention and effect:
The detection method of the present invention and other cancer of the stomach relatively has the following advantages:
The first, the present invention's a plurality of characteristic proteins of adopting patients with gastric cancer and normal person to have difference combine and carry out detection to cancer of the stomach serum, the mass spectra model that provides is the new method and the new way of cancer of the stomach early detection and examination, and for finding that further new cancer of the stomach biological marker provides the foundation;
The second, relatively having higher susceptibility and specificity with in the past serology detection method, is a kind of detection on the protein science level, provides new standard to the early detection of cancer of the stomach;
Three, the present invention is because the bonded area of magnetic bead surfaces is far longer than protein chip, thereby the sensitivity of magnetic bead is higher and had higher sensitivity than protein chip, thereby optimized mass spectra model;
Four, the present invention initiates and has adopted the regulation and control of quantitative property spectrum, and CV＜5～10% make the threshold peak average M of magnetic bead more reliable and accurate than protein chip;
Five, the present invention initiates and has adopted with mass spectral standardization quality controlled serum (slurry), mark 2746 ± 1Da, 5909 ± 1Da, 6634 ± 1Da base peak (molecular weight) quality control standard qualitatively in the mass spectrum with being used in the standardization quality controlled serum (slurry), make molecular weight error＜0.01%, thus the accurate protein spectrum collection of illustrative plates that obtains;
Six, the construction method of mass spectra model of the present invention design accurately and rationally feasible provides the appraisal procedure of a kind of new examination and recurrence, transfer for the case fatality rate that reduces China's cancer of the stomach, the clinical cure that improves cancer of the stomach;
Seven, utilize the present invention to analyze 180 parts of serum specimens with double-blind study, its susceptibility of differentiating patients with gastric cancer and normal person is 96%, and specificity is 91%; Cancer of the stomach and stomach benign disease patient susceptibility 93%, specificity 91%, patients with gastric cancer and lymph nodal metastasis susceptibility 87%, specificity 93%; Patients with gastric cancer and cancer of the stomach far-end shift susceptibility 81%, specificity 93%.
Therefore the present invention can realize cancer of the stomach is carried out early warning, early detection.
Figure of description
Fig. 1 cancer of the stomach and normal human serum polypeptide mass-spectrogram
Fig. 2 cancer of the stomach and normal human serum protein spectrum collection of illustrative plates
The mass spectra model that Fig. 3 patients with gastric cancer and normal person differentiate
The mass spectra model that Fig. 4 patients with gastric cancer and stomach benign disease are differentiated
Fig. 5 patients with gastric cancer and lymph nodal metastasis and cancer of the stomach far-end shift the mass spectra model of differentiating
M/z represents the mass-to-charge ratio of differential protein among the figure, and M represents the threshold peak average of this differential protein
Embodiment
The present invention will be described further in conjunction with specific embodiments, and these examples only are used for illustration purpose, and are not used in the restriction scope of the invention.
Embodiment 1 differentiation and mass spectral kit normal and patients with gastric cancer prepares
(1) experimental technique
Serum before 66 routine patients with gastric adenocarcinoma (wherein I phases 14 example, II phases 20 example, III phases 14 example, IV phases 16 example, 43～79 years old age, The median age 52 years old) and 24 routine stomach benign disease patients' (42～78 years old age, The median age 51 years old) the art.90 control serums derive from liver function, renal function etc. and check all normal health check-up crowd from healthy volunteer (40～69 years old age, The median age 49 years old).The person under inspection gathers venous blood 1mL on an empty stomach, left standstill 2 hours in 4 ℃ of refrigerators immediately after the collection, centrifugal 10 minutes separation of serum of 4 ℃ of 4000r/min, with serum in centrifugal once more 5 minutes of 4 ℃ of 12000r/min, remove all residual cell fragment and insolubless, on ice serum is packed as 100 μ L/ pipe, totally 5 pipes are stored in-80 ℃ of refrigerators.Avoid multigelation.
The magnetic bead operation steps
Blood serum sample is handled: from-80 ℃ of refrigerators, take out serum, and in 4 ℃, the centrifugal 5min of 10000rpm.Get 10 μ L blood serum samples, add 20 μ L U9 treating fluid (9mol/L urea, 2%CHAPS, 1%DTT, 50mmol/L Tris-CL, pH9.0), fully mixing takes out behind the ice bath vibration 30min, add 360 μ L binding buffer liquid (100mmol/L NaAc, pH4.0), mixing immediately.
Sample and wash-out on the magnetic bead: the sample 100 μ L that handle well are added in the PCR pipe that installs WCX magnetic bead (weak anionic surface, hydroxy-acid group are caught positive charge group albumen), put and hatch 30min on the magnetic processor, remove liquid.Add 100 μ L magnetic bead binding buffer liquid (50mmol/L NaAc, pH 4.0～4.3) to the PCR pipe that installs the WCX magnetic bead, put and hatch 2 minutes on the magnetic processor, remove liquid, repeat aforesaid operations 2 times.Add 10 μ L Elution Buffer 2min, the wash-out sample is to supernatant.Get 5 μ L supernatants and move in another PCR pipe, add the abundant mixing of 5 μ L SPA saturated solutions, get 1 μ L mixed solution application of sample on Au or Steel sheet, dry the back and go up the machine measurement.
Data aggregation
The Au that handles well or Steel sheet are inserted MALDI-TOF-MS carry out the protein spectrum analysis.Before reading of data, proofread and correct mass spectrometer with the chip that is added with the all-in-one standard protein, make molecular weight error＜0.1%.The major parameter of reading apparatus is set in this research, and the highest detection molecular weight is 50kDa, optimizes molecular weight ranges 1000～15000Da, optimal accumulated center 8000Da, and data acquisition parameters scope 20～80, collection adds up to 130 times.Set in the software and read the sheet program, with reading of data.Quantitatively property control and mass spectrum laser energy regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 6634.0Da intensity transfers to 50% mass signal intensity in the standardization quality controlled serum (slurry) with mass spectral standardization quality controlled serum (slurry).With 2746 ± 1Da, 5909 ± 1Da, 6634 ± 1Da base peak is demarcation property (molecular weight) quality control standard in the mass spectrum, makes molecular weight error＜0.01%, thus the accurate protein spectrum collection of illustrative plates that obtains.
Statistical analysis
All peak spectrums of application software analyzing and processing form the protein spectrum collection of illustrative plates.All collection of illustrative plates have all carried out standardization, and are unified to they own whole population of ions (summation of peak area).The maximal value that quantitative standards peak 6634.0Da intensity transfers to 40～50% signal intensities will be used in the standardization quality controlled serum, and proofread and correct with the most significant peak, then carried out in " minimizing baseline " definition protein peak (s/n＞5, minimum peak intensity＞1.6) again.Analyze the protein peak between all 1～50kDa, and scrutiny each corresponding peak, calculate the mean value M at peak, the standard error (SD) and the coefficient of variation (CV%).Check the relatively protein peak of paired sample with Mann-Whitney U, calculate the P value.
Cancer of the stomach detects the polypeptide protein screening
Check the relatively protein peak of paired sample, CV＜5～10% with Mann-Whitney U; Patients with gastric cancer and normal population that initial analysis filters out P＜0.00001 have 10 difference polypeptide proteins, and the expression variation of the molecular weight at different peaks, threshold peak average M and characteristic protein sees Table one:
The expression of table one patients with gastric cancer characteristic protein changes
The expression of molecular weight of albumen (m/z) patients with gastric cancer albumen threshold peak characteristics of mean albumen changes
M±SD
1465 10.21±6.29 ↑
5089 8.29±6.27 ↑
5916 7.31±5.93 ↑
8892 11.56±8.33 ↑
11471 13.23±12.22 ↑
11680 21.64±17.12 ↑
8936 10.71±8.31 ↑
17250 3.71±3.05 ↓
6443 2.76±2.81 ↓
8696 2.87±1.45 ↓
In the table one: to upward arrow " ↑ " is the rise characteristic protein of high expressed in patients with gastric cancer serum; Arrow " ↓ " is a low downward modulation characteristic protein of expressing in patients with gastric cancer serum downwards;
Fig. 1,2 is the raw mass spectrum collection of illustrative plates at 3 differential protein peaks in cancer of the stomach and the normal human serum (8892,11471 and 11680m/z); Last 1 collection of illustrative plates (CANCER) is a cancer of the stomach, and following 1 collection of illustrative plates (NORMAL) is a normal healthy people.
With the characteristic protein of above-mentioned 10 differential proteins as cancer of the stomach mass spectrum inspection model;
Embodiment 2 clinic trial and double-blind
Because combining just, cancer of the stomach and normal person etc. can be separated fully a plurality of characteristic proteins, so choose any two or more albumen in above-mentioned 10 characteristic proteins, according to the mass-to-charge ratio m/z value of each protein peak and with the threshold peak average M of this albumen, set up patients with gastric cancer and normal person, stomach benign disease, lymph nodal metastasis and cancer of the stomach far-end and shifted the serum characteristic protein detection mass spectra model (Fig. 3-5) that the patient differentiates in twos, said specific protein mass-to-charge ratio m/z and threshold peak average M are respectively m/z=1465, M 〉=10.21; M/z=5089, M 〉=8.29; M/z=11471, M 〉=13.23; M/z=6443, M≤2.76; M/z=1465, M 〉=16.77; M/z=8936, M 〉=10.71; M/z=11680, M 〉=21.64; M/z=8892, M 〉=11.56; Wherein the mass spectra model A that differentiates of patients with gastric cancer and normal person is by m/z=1465, M 〉=10.21; M/z=5089, M 〉=8.29 draws and forms, and the mass spectra model B of patients with gastric cancer and the discriminating of stomach benign disease is by m/z=11471, M 〉=13.23; M/z=6443, M≤2.76 are drawn and are formed, and patients with gastric cancer and lymph nodal metastasis and cancer of the stomach far-end shift the mass spectra model C of discriminating by m/z=1465, M 〉=16.77; M/z=8936, M 〉=10.71; M/z=11680, M 〉=21.64; M/z=8892, M 〉=11.56 are drawn and are formed; Molecular weight (m/z) error＜0.01%, CV＜5～10% of threshold peak average M.Utilize this mass spectra model,, just can be used for the cancer of the stomach check as long as the mass-to-charge ratio of respective egg white matter in person under inspection's serum and the threshold peak average M and the mass spectra model of the present invention of this albumen are analyzed one by one.
Utilize this mass spectra model A-C (Fig. 3-5), the mass-to-charge ratio of respective egg white matter in person under inspection's serum and the threshold peak average M and the mass spectra model of the present invention of this albumen are analyzed one by one, through clinic trial and double-blind, its susceptibility of differentiating patients with gastric cancer and normal person is 96%, and specificity is 91%; Cancer of the stomach and stomach benign disease patient susceptibility 93%, specificity 91%, patients with gastric cancer and lymph nodal metastasis susceptibility 87%, specificity 93%; Patients with gastric cancer and cancer of the stomach far-end shift susceptibility 81%, specificity 93%.
Utilize the experimental result of C8 and C18 hydrophobic matrix magnetic bead consistent with the experimental result of above-mentioned WCX anionic substrates magnetic bead.
All documents of mentioning in the present invention are incorporated by reference in this application all, is just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (3)
1. a mass spectra model that detects the cancer of the stomach serum characteristic protein is characterized in that: filter out 7 upregulated proteins and 3 down-regulation proteins as characteristic protein from serum; Choose any two or more albumen in above-mentioned 10 characteristic proteins, according to the mass-to-charge ratio m/z value of each protein peak and with the threshold peak average M of this albumen, set up patients with gastric cancer and normal person, stomach benign disease, lymph nodal metastasis and cancer of the stomach far-end and shifted the serum characteristic protein detection mass spectra model that the patient differentiates in twos; Wherein the mass spectra model A that differentiates of patients with gastric cancer and normal person is by m/z=1465, M 〉=10.21; M/z=5089, M 〉=8.29 draws and forms, and the mass spectra model B of patients with gastric cancer and the discriminating of stomach benign disease is by m/z=11471, M 〉=13.23; M/z=6443, M≤2.76 are drawn and are formed, and patients with gastric cancer and lymph nodal metastasis and cancer of the stomach far-end shift the mass spectra model C of discriminating by m/z=1465, M 〉=16.77; M/z=8936, M 〉=10.71; M/z=11680, M 〉=21.64; M/z=8892, M 〉=11.56 are drawn and are formed.
2, a kind of preparation method who detects cancer of the stomach haemocyanin mass spectra model may further comprise the steps:
1) 4 ℃ of conditions are collected the patients with gastric cancer serum of clarifying a diagnosis through pathology and the serum that is defined as normal healthy person through health check-up as two groups of serum specimens in following 2 hours, and it is standby to carry out-80 ℃ of cryogenic freezings;
2) adopt the WCX magnetic bead that the albumen of two groups of serum specimens of described patients with gastric cancer and normal person is adsorbed;
3) use the mastrix-assisted laser desorption ionization time of flight mass spectrometer, with nitrogen laser (337nm) and 80cm or 120cm tof tube analysis array, or, obtain two histone mass-spectrograms thus with liquid chromatography mass combined instrument (LC-MS) the WCX magnetic bead that is combined in the weak anionic surface or two groups of haemocyanins on C8 and the C18 hydrophobic matrix magnetic bead being read behind the haemocyanin of electron spray ionisation wash-out;
4) before each experimental data is collected, rectify an instrument with standard protein and mass spectral standardization Quality Control O type serum, make CV＜5～10% of protein molecular weight error＜0.01% and threshold peak average M, and collect the serum mass-spectrogram data that patients with gastric cancer and normal person optimize accurately, quantitatively;
5) the gained data are carried out statistical procedures, difference according to protein peak between patients with gastric cancer and the normal human serum, detecting between 2 groups of haemocyanin mass spectrogram spectrums has 10 stable differential proteins and threshold peak thereof, and 7 upregulated proteins and 3 down-regulation proteins are as characteristic protein; With the characteristic protein of described 10 differential proteins as cancer of the stomach protein spectrum model;
6) choose any two or more albumen in described 10 characteristic proteins, be configured for detecting cancer of the stomach serum characteristic protein mass spectra model with the mass-to-charge ratio m/z value of each protein peak and with the threshold peak average M of this albumen; Wherein said specific protein mass-to-charge ratio m/z and threshold peak average M are respectively m/z=1465, M 〉=10.21; M/z=5089, M 〉=8.29; M/z=11471, M 〉=13.23; M/z=6443, M≤2.76; M/z=1465, M 〉=16.77; M/z=8936, M 〉=10.71; M/z=11680, M 〉=21.64; M/z=8892, M 〉=11.56.
3, choose any two or more characteristic proteins in 10 albumen of serum as claimed in claim 1, utilize this mass spectra model, the mass-to-charge ratio of respective egg white matter in person under inspection's serum and the threshold peak average M and the mass spectra model of the present invention of this albumen are analyzed one by one, just can be applicable to early detection, examination and the recurrence of cancer of the stomach, the assessment of transfer.
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