CN1455257A - Method of diagnosing lung cancer using surface modified protein chip - Google Patents
Method of diagnosing lung cancer using surface modified protein chip Download PDFInfo
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- CN1455257A CN1455257A CN 03136951 CN03136951A CN1455257A CN 1455257 A CN1455257 A CN 1455257A CN 03136951 CN03136951 CN 03136951 CN 03136951 A CN03136951 A CN 03136951A CN 1455257 A CN1455257 A CN 1455257A
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Abstract
First, by using blood serums of lung cancer patients and health persons prepares the ionized time-of-flight mass spectrum protein chips with surface enhanced laser desorption. The protein spectrums of the chips are measured. Six labeled differentia proteins are screened out from the protein spectrum as the symbol proteins of lung cancer. Minim blood serum taken from the patient to be tested is used to prepare the ionized time-of-flight mass spectrum protein chips with surface enhanced laser desorption and its protein spectrums are measured. Based on the intensity of peak value, using the protein with molecular weight being as 5910 Dalton and 1030 Dalton judges whether the patient is the lung cancer patient. The sensitivity and the specificity of the invented diagnostic method are better than any unimolecule index utilized in clinic. The invention possesses the advantages of easy sampling, intact, little dosage (5-10 micro liter), easy and fast operation.
Description
Technical field the present invention relates to a kind of method of lung cancer diagnosis of utilizing the finishing protein chip, belongs to the proteomics applied technical field.
Background technology lung cancer is one of modal malignant tumour in various places, the world today.Calendar year 2001, there were 169500 routine lung cancer new patients in the U.S., died from 157400 people of lung cancer, accounted for more than 1/3 of the total death toll of cancer.The many big cities of China over nearly 30 years the lung cancer morbidity rate obviously rise, accounted for the first place of common cancer.And may obtain diagnosis hardly in early days.Chang Gui inspection is mainly according to iconography method such as X-ray and computed tomography (CT) clinically.Though marker for lung cancer is maximum in the kinds of tumor mark, comprising protein, endocrine substance, enzyme, peptide class and various antigenic substance.But generally speaking all lack specificity, can only be as the reference index of observing change of illness state.Therefore seek and establish the high marker molecule of lung cancer high specificity, susceptibility and seem particularly important as the early diagnosis or the early warning of lung cancer.
Protein group is the biotechnology that rises rapidly in the world behind genome, and one of its main application fields is medical diagnosis on disease.Any in theory disease is before pathological change occurring, and intracellular protein is at composition and quantitatively all corresponding change can be arranged.By to protein dynamic " making an inventory ", find small but significant variation, be the new way that quilt is spoken highly of of carrying out disease early diagnosis and early warning.Certainly, realize the prerequisite of this diagnosis, will find out exactly the representational distinctive mark protein molecular of various diseases (Biomarker).Surface enhanced laser desorption ionization flight time mass spectrum (Surfaced Enhanced Laser Desorption/Ionization; SELDI) protein biochip technology is the proteomic techniques that new development is got up.Except characteristics such as quick, amount of samples is few, can also directly use biological sample with complicated protein ingredient.Since at the bottom of calendar year 2001, adopted this technology from serum, to obtain successively the representational marker molecule of colorectal cancer, breast cancer, oophoroma and prostate cancer in the world.Still there is not the report that adopts the SELDI technology from serum, to obtain the lung cancer marker molecule in the world so far.
Summary of the invention the objective of the invention is to propose a kind of finishing protein chip that utilizes and carries out method of lung cancer diagnosis, utilize the SELDI protein biochip technology of recent development, find out the significant difference of the protein spectrum of lung cancer patient serum and normal human serum, again the protein spectrum in the case serum to be measured is carried out determination and analysis, thereby to differentiate case automatically, intuitively.
The finishing protein chip that utilizes that the present invention proposes carries out method of lung cancer diagnosis, may further comprise the steps:
(1) gets the lung cancer patient made a definite diagnosis and normal person's serum, be prepared into surface enhanced laser desorption ionization flight time mass spectrum protein-chip, measure its protein spectrum, filtering out molecular weight from protein spectrum is that 5910 dalton, 5931 dalton, 5342 dalton, 6116 dalton, 11510 dalton and 1030 daltonian six sign differential proteins are as lung cancer marker protein matter;
(2) get patient's trace serum to be measured, be prepared into surface enhanced laser desorption ionization flight time mass spectrum protein-chip, measure its protein spectrum;
(3) the above-mentioned patient's to be measured of analysis protein spectrum, if occur six lung cancer marker protein matter in above-mentioned the 1st step on the protein spectrum, be that 5910 daltonian protein are judged further then with molecular weight, if the intensity at this peak is greater than 7.059, then this patient to be measured is the lung cancer positive, if the intensity at this peak is less than or equal to 7.059, then this patient to be measured is the lung cancer feminine gender;
(4) above-mentioned the 3rd step being judged as the patient to be measured of lung cancer feminine gender, is that 1030 daltonian protein are judged with molecular weight again, if the intensity at this peak is greater than 33.261, then this patient to be measured heavily divides the lung cancer positive into.
The process of preparation surface enhanced laser desorption ionization flight time mass spectrum protein-chip may further comprise the steps in the said method:
(1) get fresh respectively or-20 degrees centigrade to-196 degrees centigrade test serums of preserving down, add urea in serum, serum and urea volume ratio are 1: 1.5～1: 3, mix vibration 15～30 minutes, and be standby;
(2) be added drop-wise to the stand-by sampling point surface that adds of weak cation exchange chip with watery hydrochloric acid, kept 5～10 minutes, and, respectively adding Dropwise 5 on the sampling point～50 microlitres, 100 mM sodium acetate buffers then with high-purity washing chip three times, hatch under the room temperature, discard sodium acetate buffer after 5～15 minutes;
(3) add serum/urea admixture and the sodium acetate buffer that adds above-mentioned first step preparation on the sampling point respectively at each, the ratio that adds is: serum/urea admixture: sodium acetate buffer=1: 8～1: 20, vibration was hatched 15～30 minutes, discard the liquid that adds on the sampling point, receive washing chip 2～3 times with 50～200 microlitres, 100 mM acetate, each 3～5 minutes, chip washs 1～3 time with high purity water then, after treating that chip surface dries naturally, respectively add the sinapic acid that the sampling point surface adds 1～2 time 0.5 microlitre, chip surface dries naturally;
(4) measuring each molecular weight that adds contained range protein on the sampling point and relative populations thereof is intensity, and sets up relation between the two, i.e. protein spectrum.
The finishing protein chip that utilizes that the present invention proposes carries out method of lung cancer diagnosis, with it clinical 39 routine lung cancer patients of obtaining and 35 routine normal human serums are carried out preliminary blind sieve, the result shows, its susceptibility that lung cancer is detected is 92.3%, specificity is 97.1%, (susceptibility is defined as the number percent that detects positive patient's number and whole pulmonary carcinosis numbers, specificity is defined as expresses negative normal number and whole normally number percents of numbers), this shows that its diagnostic function is good, is better than any individual molecule index in the present clinical use.And have collection of specimens simple, do not have advantages such as infringement, consumption few (5-10 microlitre), technical operation be easy, quick.
Description of drawings
Fig. 1 is the interior peak-shaped curve example of 5000-8000 dalton molecule weight range on the WCX-2 chip.Horizontal ordinate is a molecular weight among the figure, and ordinate is an intensity, above three curves be the normal person, below three curves be lung cancer patient, the protein peak that marks among the figure is compared discrepant protein peak for patient with the normal person.
Embodiment: the finishing protein chip that utilizes that the present invention proposes carries out method of lung cancer diagnosis, at first get the serum of having made a definite diagnosis lung cancer patient and normal person, be prepared into surface enhanced laser desorption ionization flight time mass spectrum protein-chip, measure its protein spectrum, from protein spectrum, filter out molecular weight and be 5910 dalton, 5931 dalton, 5342 dalton, 6116 dalton, 11510 dalton and 1030 daltonian six with the discrepant protein of normal person as lung cancer marker protein matter.This diagnostic method has obtained lung cancer patient that strictness makes a definite diagnosis to 75 and 66 healthy people's blood serum sample is analyzed.The lung cancer patient blood serum sample is from the Capital University of Medical Sciences's attached Tiantan Hospital division of respiratory disease, and all patients all have complete clinical data and pathological diagnosis, wherein adenocarcinoma of lung 32 examples, phosphorus cancer 22 examples, small-cell carcinoma of the lung 21 examples.Patient age is at 40-77 between year.The sample that 66 routine control group serum filter out when deriving from General Administration of Sport's health screening, age and sex and lung cancer patient mate substantially.Get patient's trace serum to be measured, be prepared into surface enhanced laser desorption ionization flight time mass spectrum protein-chip, measure its protein spectrum; Analyze patient's to be measured protein spectrum, if occur six lung cancer marker protein mass peaks on the protein spectrum, be that 5910 daltonian protein are judged further then with molecular weight, if the intensity at this peak is greater than 7.059, then this patient to be measured is the lung cancer positive, if the intensity at this peak is less than or equal to 7.059, then this patient to be measured is the lung cancer feminine gender; To being judged as the patient to be measured of the lung cancer positive, be that 1030 daltonian protein are judged with molecular weight again, if the intensity at this peak is greater than 33.261, then this patient to be measured is the lung cancer positive.
The process of preparation surface enhanced laser desorption ionization flight time mass spectrum protein-chip is in the said method: get fresh respectively or-20 degrees centigrade to-196 degrees centigrade test serums of preserving down, in serum, add urea, serum and urea volume ratio are 1: 1.5～1: 3, mix vibration 15～30 minutes, standby; Be added drop-wise to the stand-by sampling point surface that adds of weak cation exchange chip with watery hydrochloric acid, kept 5～10 minutes, and, respectively adding Dropwise 5 on the sampling point～50 microlitres, 100 mM sodium acetate buffers then with high-purity washing chip three times, hatch under the room temperature, discard sodium acetate buffer after 5～15 minutes; Add at each and to add serum/urea admixture and sodium acetate buffer on the sampling point respectively, the ratio that adds is: serum/urea admixture: sodium acetate buffer=1: 8～1: 20, vibration was hatched 15～30 minutes, discard the liquid that adds on the sampling point, receive washing chip 2～3 times with 50～200 microlitres, 100 mM acetate, each 3～5 minutes, chip washs 1～3 time with high purity water then, after treating that chip surface dries naturally, respectively add the sinapic acid that the sampling point surface adds 1～2 time 0.5 microlitre, chip surface dries naturally; Measuring each molecular weight that adds contained range protein on the sampling point and relative populations thereof is intensity, and sets up relation between the two, i.e. protein spectrum.
In the inventive method, used surface enhanced laser desorption ionization flight time mass spectrum (Surfaced EnhancedLaser Desorption/Ionization; SELDI) protein biochip technology at first is the surface chemical modification in addition to chip, makes it can catch the protein (as hydrophobicity, metal compatibility or certain limit isoelectric point etc.) that has a certain characteristic in the sample specifically.Protein molecule is ionized under the laser beam bombardment, and flies to detecting device under electric field acceleration and high vacuum.Its flying speed depends on molecular mass, can read the molecular weight of every kind of protein according to the flight time of accurate mensuration, so be called flight time mass spectrum.
Introduce embodiments of the invention below.
Embodiment 1: sample collection: patient to be detected, 5 milliliters of extracting vein bloods, not anti-freezing.Deposited 30 minutes for 4 degrees centigrade, per minute 4000 left the heart 15 minutes, got-80 degrees centigrade of preservations after the packing of serum equivalent.Get patients serum's 10 microlitres of-80 degrees centigrade of preservations, in serum, add the urea of 30 microlitres, mix vibration 30 minutes.Be added drop-wise to the 1st to the 8th of weak cation exchange chip (its model is WCX-2) with the watery hydrochloric acid of 5 microlitres and add sampling point (A-H point) surface 5 minutes, with high-purity washing chip three times, each adds and adds 50 microlitres, 100 mM sodium acetate buffers on the sampling point at chip, hatched under the room temperature 15 minutes, and discarded sodium acetate buffer.Add sampling point at each and add above-mentioned serum/urea admixture of 10 microlitres and 90 microlitres, 100 mM sodium acetate buffers respectively, after vibration is hatched 30 minutes, discard the liquid that adds on the sampling point.Receive washing chip 3 times with 150 microlitres, 100 mM acetate, each 5 minutes, then chip was with high purity water washing 3 times, treat that chip surface dries naturally after, respectively add the sinapic acid (SPA) that the sampling point surface adds 2 times 0.5 microlitres, chip surface dries naturally.On adding the sample of sampling point, each gathers molecular weight data with the protein-chip reading machine, the protein-chip reading machine is provided with as follows: laser intensity 190, detector sensitivity 9, optimize molecular weight range 3000 to 30000 atomic weight units, highest weight 200000 atomic weight units, each adds on the sampling point collects the molecule measuring given data 85 times, adopts Sai Fuji special software 3.0 (the Ciphergen proteinchip 3.0) of company to carry out data processing and statistical analysis.Specifically be provided with as follows during software analysis: signal to noise ratio (S/N ratio) " signal/noise " is 5, collecting threshold value " minimum peak threshold " is 10%, aggregate quality " cluster mass " is 2%, and aggregate quality compensation signal to noise ratio (S/N ratio) " the signal/noise for the secondpass " is 2%.The gained data are with the marker molecule combination style analysis software (BiomarkerPattern 4.0) of Sai Fuji company special use, look into that molecular weight is that 5910 daltonian peak intensities are 11.066 in the gained data, are diagnosed as the lung cancer positive.
Embodiment 2: collect 10 milliliters of venous patient blood to be detected, not anti-freezing.Room temperature was placed 10 minutes, and per minute 3000 left the heart 30 minutes, got after the packing of serum equivalent-196 degrees centigrade and preserved.Get-196 degrees centigrade of patients serum's 5 microlitres extremely, in serum, add the urea of 10 microlitres, mix vibration 15 minutes.Be added drop-wise to the 1st to the 8th of weak cation exchange chip (WCX-2 chip) with the watery hydrochloric acid of 10 microlitres and add sampling point (A-H point) surface 8 minutes, with high-purity washing chip three times.Each adds chip sampling point and adds 10 microlitres, 100 mM sodium acetate buffers, hatches under the room temperature 5 minutes, discards sodium acetate buffer.Add sampling point at each and add above-mentioned serum/urea admixture of 5 microlitres and 95 microlitres, 100 mM sodium acetate buffers respectively, vibration was hatched 20 minutes, discard the liquid that adds on the sampling point, receive washing chip 3 times with 100 microlitres, 100 mM acetate, each 3 minutes, then chip was with high purity water washing 2 times, treat that chip surface dries naturally after, respectively add the sinapic acid (SPA) that the sampling point surface adds 1 time 1 microlitre, chip surface dries naturally.On adding the sample of sampling point, each gathers molecular weight data with the protein-chip reading machine, the protein-chip reading machine is provided with as follows: laser intensity 205, detector sensitivity 8, optimize molecular weight range 5000 to 50000 atomic weight units, highest weight 200000 atomic weight units, each adds on the sampling point collects the molecule measuring given data 65 times, adopts Sai Fuji company special software 3.0 (Ciphergenproteinchip 3.0) to carry out data processing and statistical analysis.Specifically be provided with as follows during software analysis: signal to noise ratio (S/N ratio) " signal/noise " is 3, collecting threshold value " minimum peak threshold " is 10%, aggregate quality " cluster mass " is 0.5%, and aggregate quality compensation signal to noise ratio (S/N ratio) " the signal/noise for the second pass " is 2%.The gained data make up style analysis software (Biomarker Pattern 4.0) with the marker molecule of Sai Fuji company special use, molecular weight 5910 daltonian peak intensities are 5.731 in the data, molecular weight is that 1030 daltonian intensity are 9.303, shows automatically to be diagnosed as the lung cancer feminine gender.
Embodiment 3: patient to be detected, 5 milliliters of extracting vein bloods, not anti-freezing.Deposited 30 minutes for 4 degrees centigrade, per minute 4000 left the heart 15 minutes, got-80 degrees centigrade of preservations after the packing of serum equivalent.Get patients serum's 4 microlitres of-80 degrees centigrade of preservations, in serum, add the urea of 6 microlitres, mix vibration 20 minutes.Be added drop-wise to the 1st to the 8th of weak cation exchange chip (WCX-2 chip) with the watery hydrochloric acid of 5 microlitres and add sampling point (A-H point) surface 5 minutes, with high-purity washing chip three times, each adds and adds 50 microlitres, 100 mM sodium acetate buffers on the sampling point at chip, hatched under the room temperature 15 minutes, and discarded sodium acetate buffer.Add sampling point at each and add above-mentioned serum/urea admixture of 5 microlitres and 60 microlitres, 100 mM sodium acetate buffers (serum/urea admixture is 1: 12 with the ratio of 100 mM sodium acetate buffer volumes) respectively, after putting into wet box and hatching 20 minutes, discard the liquid that adds on the sampling point.Receive washing chip 3 times with 5 microlitres, 100 mM acetate, each 5 minutes, then chip was with high purity water washing 3 times, treat that chip surface dries naturally after, respectively add the sinapic acid (SPA) that the sampling point surface adds 2 times 0.5 microlitres, chip surface dries naturally.On adding the sample of sampling point, each gathers molecular weight data with the protein-chip reading machine, the protein-chip reading machine is provided with as follows: laser intensity 195, detector sensitivity 9, optimize molecular weight range 3000 to 30000 atomic weight units, highest weight 200000 atomic weight units, each adds on the sampling point collects the molecule measuring given data 85 times, adopts Sai Fuji company special software 3.0 (Ciphergenproteinchip 3.0) to carry out data processing and statistical analysis.Specifically be provided with as follows during software analysis: signal to noise ratio (S/N ratio) " signal/noise " is 5, collecting threshold value " minimum peak threshold " is 10%, aggregate quality " cluster mass " is 2%, and aggregate quality compensation signal to noise ratio (S/N ratio) " the signal/noise for the second pass " is 2%.Molecular weight is that 5910 daltonian peak intensities are 10.546 in the gained data, but molecular weight 1030 daltonian peak intensities are 39.336, are diagnosed as the lung cancer positive.
Claims (2)
1, a kind of finishing protein chip that utilizes carries out method of lung cancer diagnosis, it is characterized in that this method may further comprise the steps:
(1) gets the lung cancer patient made a definite diagnosis and normal person's serum, be prepared into surface enhanced laser desorption ionization flight time mass spectrum protein-chip, measure its protein spectrum, filtering out molecular weight from protein spectrum is that 5910 dalton, 5931 dalton, 5342 dalton, 6116 dalton, 11510 dalton and 1030 daltonian six sign differential proteins are as lung cancer marker protein matter;
(2) get patient's trace serum to be measured, be prepared into surface enhanced laser desorption ionization flight time mass spectrum protein-chip, measure its protein spectrum;
(3) the above-mentioned patient's to be measured of analysis protein spectrum, if occur six lung cancer marker protein mass peaks in above-mentioned the 1st step on the protein spectrum, be that 5910 daltonian protein are judged further then with molecular weight, if the intensity at this peak is greater than 7.059, then this patient to be measured is the lung cancer positive, if the intensity at this peak is less than or equal to 7.059, then this patient to be measured is the lung cancer feminine gender;
(4) above-mentioned the 3rd step being judged as the patient to be measured of lung cancer feminine gender, is that 1030 daltonian protein are judged with molecular weight again, if the intensity at this peak is greater than 33.261, then this patient to be measured is the lung cancer positive.
2, the method for claim 1 is characterized in that the process that wherein prepares surface enhanced laser desorption ionization flight time mass spectrum protein-chip may further comprise the steps:
(1) get fresh respectively or-20 degrees centigrade to-196 degrees centigrade test serums of preserving down, add urea in serum, serum and urea volume ratio are 1: 1.5～1: 3, mix vibration 15～30 minutes, and be standby;
(2) be added drop-wise to the stand-by sampling point surface that adds of weak cation exchange chip with watery hydrochloric acid, kept 5～10 minutes, and, respectively adding Dropwise 5 on the sampling point～50 microlitres, 100 mM sodium acetate buffers then with high-purity washing chip three times, hatch under the room temperature, discard sodium acetate buffer after 5～15 minutes;
(3) add serum/urea admixture and the sodium acetate buffer that adds above-mentioned first step preparation on the sampling point respectively at each, the ratio that adds is: serum/urea admixture: sodium acetate buffer=1: 8～1: 20, vibration was hatched 15～30 minutes, discard the liquid that adds on the sampling point, receive washing chip 2～3 times with 50～200 microlitres, 100 mM acetate, each 3～5 minutes, chip washs 1～3 time with high purity water then, after treating that chip surface dries naturally, respectively add the sinapic acid that the sampling point surface adds 1～2 time 0.5 microlitre, chip surface dries naturally;
(4) measuring each molecular weight that adds contained range protein on the sampling point and relative populations thereof is intensity, and sets up relation between the two, i.e. protein spectrum.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1735620A2 (en) * | 2004-03-30 | 2006-12-27 | Eastern Virginia Medical School | Lung cancer biomarkers |
| CN1300580C (en) * | 2004-12-31 | 2007-02-14 | 中国人民解放军第306医院 | Mass spectrum model for detecting liver cancer serum characteristic protein and method for preparation |
| CN1851454B (en) * | 2005-04-22 | 2010-12-22 | 复旦大学附属中山医院 | Method for determining serum protein fingerprint |
| CN104198709A (en) * | 2008-09-09 | 2014-12-10 | 私募蛋白质体公司 | Lung cancer biomarkers and uses thereof |
| CN104777313A (en) * | 2010-07-09 | 2015-07-15 | 私募蛋白质体公司 | Lung cancer biomarkers and uses thereof |
-
2003
- 2003-05-23 CN CN 03136951 patent/CN1455257A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1735620A2 (en) * | 2004-03-30 | 2006-12-27 | Eastern Virginia Medical School | Lung cancer biomarkers |
| EP1735620A4 (en) * | 2004-03-30 | 2008-04-09 | Eastern Virginia Med School | Lung cancer biomarkers |
| CN1300580C (en) * | 2004-12-31 | 2007-02-14 | 中国人民解放军第306医院 | Mass spectrum model for detecting liver cancer serum characteristic protein and method for preparation |
| CN1851454B (en) * | 2005-04-22 | 2010-12-22 | 复旦大学附属中山医院 | Method for determining serum protein fingerprint |
| CN104198709A (en) * | 2008-09-09 | 2014-12-10 | 私募蛋白质体公司 | Lung cancer biomarkers and uses thereof |
| CN104777313A (en) * | 2010-07-09 | 2015-07-15 | 私募蛋白质体公司 | Lung cancer biomarkers and uses thereof |
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