CN108152430A - Oophoroma marker detection kit and detection method based on PRM detections - Google Patents

Oophoroma marker detection kit and detection method based on PRM detections Download PDF

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Publication number
CN108152430A
CN108152430A CN201711396031.3A CN201711396031A CN108152430A CN 108152430 A CN108152430 A CN 108152430A CN 201711396031 A CN201711396031 A CN 201711396031A CN 108152430 A CN108152430 A CN 108152430A
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human
heavy
prm
oophoroma
light
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陈薇
阮宏强
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Shanghai Applied Protein Technology Co Ltd
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Shanghai Applied Protein Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • G01N30/6073—Construction of the column body in open tubular form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Abstract

The present invention provides a kind of oophoroma marker detection kit and its detection method based on PRM detections, belong to oophoroma marker detection technical field, for quick and precisely detecting the content of 5 kinds of markers, it includes conventional Mass Spectrometer Method reagents, heavy label standard peptide fragment test tube and dedicated chromatographic column, by 10 artificial synthesized isotope peptide fragments of mark again that 5 ovarian cancer protein markers are mixed into serum, absolute quantification analysis is carried out to 5 ovarian cancer protein markers in Inner sources in serum using PRM technologies, to achieve the purpose that help the diagnosis of oophoroma, with easy to operate, it takes few, experimental period is short, the advantages of high specificity and high sensitivity.

Description

Oophoroma marker detection kit and detection method based on PRM detections
Technical field
The invention belongs to oophoroma marker detection technical field more particularly to a kind of oophoroma marks based on PRM detections Will analyte detection kit further relates to a kind of detection method using the kit.
Background technology
Malignant tumor of ovary is one of common malignant tumour of female sex organ, and incidence is only second to cervix cancer and son Carcinoma of corpus uteri and arrange and occupy third position, account for about the 4% of women whole body malignant tumour.But because oophoroma causes the dead, but account for all kinds of gynaecology and swell Serious threat is caused in the first place of knurl to women's life.
Parallel reaction monitoring (Parallel reaction monitoring, PRM) parallel reaction monitoring belongs to targeting MS/ MS is analyzed, and constantly whole fragment ion collection of illustrative plates of each target parent ion can be remembered in entire liquid phases separation Record.Compared to the pattern that traditional SRM only detects object ion pair, PRM detects all fragments in selected parent ion window Information.The main advantages of PRM are exactly to have used the Orbitrap mass analyzers of ultrahigh resolution, can by interference information with True signal distinguishes and can preferably ensure compared to traditional SRM methods the selectivity of analysis.
The OVA1 testing cassetes of Vermillion company of the U.S. and Quest Diagnostics companies R ＆ D Cooperation can be in pelvic cavity It checks whether suffer from oophoroma in mass, and determines the need for operative treatment, patient and medical staff is helped to determine to belong to What kind of operation and should implementation be responsible for by whom.Five immunoassays are combined in the qualitative serologic test of OVA1 oophoromas Form a single numerical scoring system.The immunoassay determines five effective biomarkers --- thyroxine, APoA-I, β2-microglobulin (Beta2M), transferrins and cancer antigen 125, for determining that pelvic lump women may Tumorigenic possibility.But the detection of OVA1 testing cassetes it is complicated for operation, time-consuming, and its sensitivity and specificity is relatively Difference cannot meet the needs.
Invention content
Based on the prior art there are the above problem, the present invention provides a kind of oophoroma marker detection based on PRM detections Kit and its detection method, for quick and precisely detecting the content of 5 kinds of markers, it includes conventional Mass Spectrometer Method reagent, Heavy label standard peptide fragment test tube and dedicated chromatographic column, by mixing 5 ovarian cancer protein marks into serum 10 artificial synthesized isotope peptide fragments of mark again of object, using PRM technologies to 5 ovarian cancer protein markers in Inner sources in serum Carry out absolute quantification analysis, with achieve the purpose that help oophoroma diagnosis, have it is easy to operate, take less, experimental period it is short, The advantages of high specificity and high sensitivity.
The present invention is achieved the goal by technical solution in detailed below：
A kind of chromatographic column for being applicable in oophoroma PRM detections, including column tube, filler and pipette tips, filler is filled in column tube Interior, pipette tips are sleeved on one end of column tube；The column tube length is 115-125mm, outer diameter 355-365um, internal diameter 70- 80um；The filler is the C18 reverse phase fillers of 2.5-3.5um grain sizes.
Wherein, the column tube length be 120mm, outer diameter 360um, internal diameter 75um；The filler is 3um grain sizes C18 reverse phase fillers.
A kind of oophoroma marker detection kit based on PRM detections, including conventional Mass Spectrometer Method reagent, again same position Plain label standard peptide fragment test tube, above-mentioned chromatographic column, dissolving buffer；The heavy label standard peptide fragment test tube packet Containing ten unique sequences in thyroxine, aPoA -1, β2-microglobulin (Beta2M), transferrins and cancer antigen 125 Row, wherein each albumen respectively has two sections of peptide section sequences.
Wherein, the heavy label standard peptide fragment be VEHSDLSFSK, VNHVTLSQPK, EFQLFSSPHGK, HQTVPQNTGGK、GSPAINVAVHVFR、AADDTWEPFASGK、DLATVYVDVLK、DYVSQFEGSALGK、LGIWDDFIPK And ELPLLSPPQDK.
Wherein, the amino acid of the heavy label of the heavy label standard peptide fragment is located at peptide section sequence respectively On the arginine and lysine of C- ends.
Wherein, the concentration of every standard peptide fragment is 50fmol in the heavy label standard peptide fragment test tube.
Wherein, the dissolving buffer is the aqueous solution containing 0.1% formic acid.
A kind of detection method using above-mentioned detection kit includes the following steps：
Step S10 takes heavy label standard peptide fragment test tube, adds in 90-110uL dissolving buffer thereto, shakes mixing, The centrifugation 45-75S that speed is 12000-14000g is carried out using centrifuge, takes supernatant spare；
Step S20 takes 0.08-0.12mg haemocyanins peptide hydrolysis to add in 90-110uL dissolving buffer, shakes mixing, be made Serum peptide fragment solution, it is spare；
Step S30 takes serum peptide fragment solution made of 1.8-2.2uL steps S20, and adds in 9-11uL step S10 systems thereto Into heavy label standard peptide fragment solution in, be uniformly mixed, take 3-5uL carry out chromatography column feed materials；
The setting of chromatographic isolation gradient is as follows：
PRM detection ion table settings are as follows：
M/Z Peptide L/H Protein
574.782753 VEHSDLSFSK light sp|P61769|B2MG_HUMAN
578.789852 VEHSDLSFSK heavy sp|P61769|B2MG_HUMAN
561.81693 VNHVTLSQPK light sp|P61769|B2MG_HUMAN
565.824029 VNHVTLSQPK heavy sp|P61769|B2MG_HUMAN
638.819669 EFQLFSSPHGK light sp|P02787|TRFE_HUMAN
642.826769 EFQLFSSPHGK heavy sp|P02787|TRFE_HUMAN
583.799268 HQTVPQNTGGK light sp|P02787|TRFE_HUMAN
587.806368 HQTVPQNTGGK heavy sp|P02787|TRFE_HUMAN
683.883135 GSPAINVAVHVFR light sp|P02766|TTHY_HUMAN
688.88727 GSPAINVAVHVFR heavy sp|P02766|TTHY_HUMAN
697.814781 AADDTWEPFASGK light sp|P02766|TTHY_HUMAN
701.82188 AADDTWEPFASGK heavy sp|P02766|TTHY_HUMAN
618.347728 DLATVYVDVLK light sp|P02647|APOA1_HUMAN
622.354828 DLATVYVDVLK heavy sp|P02647|APOA1_HUMAN
700.838256 DYVSQFEGSALGK light sp|P02647|APOA1_HUMAN
704.845356 DYVSQFEGSALGK heavy sp|P02647|APOA1_HUMAN
602.324056 LGIWDDFIPK light sp|Q8WXI7|MUC16_HUMAN
606.331156 LGIWDDFIPK heavy sp|Q8WXI7|MUC16_HUMAN
618.845353 ELPLLSPPQDK light sp|Q8WXI7|MUC16_HUMAN
622.852452 ELPLLSPPQDK heavy sp|Q8WXI7|MUC16_HUMAN
Mass spectrograph setting is as follows：
First mass spectrometric, scanning range：500-800m/z, resolution ratio：70000 (@m/z 200), AGC target：3e6, Maximum IT：200ms；After first mass spectrometric 15 two level PRM scannings are triggered according to ingredient lists；
Two level PRM is scanned, Isolation window：2Th, resolution ratio：35000 (@m/z 200), AGC target：3e6, Maximum IT：200ms, Microscans：1, MS2Activation Type：HCD, NCE：27；
Step S40 carries out PRM mass spectrometric datas Skyline analyses, and calculates concentration of 5 kinds of markers in Sample serum.
Wherein, each sample carries out 2-5 and repeats PRM detection experiments.
The device have the advantages that：By the present invention in that with the unique heavy isotope mark of PRM technology combination markers Remember that standard peptide carries out absolute quantification analysis to 5 kinds of albumen, to help the diagnosis to oophoroma, have it is convenient and efficient, highly selective, The advantages that high specific and high accuracy, can be with 5 kinds of oophoroma marker proteins in absolute quantification analysis serum, and detection sensitivity Height, Protein Detection is up to pg/ml levels；Using nanoliter level chromatograph Easy-nLC and high-resolution mass spectrometer Q-Exactive systems Row, analysis is more accurate, operation is simpler；Experimental period is short, and the quantitative analysis of 5 kinds of albumen can be completed in general 2 hours； Reagent contained by kit is protein and general inorganic salt solution, and no volatile, poisonous and harmful substances are safe； Absolute quantification analysis is carried out for 5 kinds of oophoroma marker proteins in serum, high specificity, experimental result consistency is high, has height The advantages that sensitivity, high specific, true and reliable using the obtained experimental result of the kit, repeatability is strong.
Description of the drawings
Fig. 1, a kind of structure diagram for the chromatographic column for being applicable in oophoroma PRM detections.
Specific embodiment
The invention will be further described in the following with reference to the drawings and specific embodiments.
Embodiment one：Chromatographic column.
A kind of chromatographic column for being applicable in oophoroma PRM detections as shown in Figure 1, including column tube (1), filler (2) With pipette tips (3), filler (2) is filled in column tube (1), and pipette tips (3) are sleeved on one end of column tube (1)；Described column tube (1) length is 120mm, outer diameter 360um, internal diameter 75um；The filler (2) is the C18 reverse phase fillers of 3um grain sizes.
Embodiment two：Oophoroma marker detection kit
A kind of oophoroma marker detection kit based on PRM detections, including conventional Mass Spectrometer Method reagent, heavy isotope mark Note standard peptide fragment test tube, above-mentioned chromatographic column, the aqueous solution containing 0.1% formic acid；The heavy label standard peptide fragment Test tube includes ten in thyroxine, aPoA -1, β2-microglobulin (Beta2M), transferrins and cancer antigen 125 Unique sequence code, wherein each albumen respectively has two sections of peptide section sequences, be respectively VEHSDLSFSK, VNHVTLSQPK, EFQLFSSPHGK、HQTVPQNTGGK、GSPAINVAVHVFR、AADDTWEPFASGK、DLATVYVDVLK、 DYVSQFEGSALGK, LGIWDDFIPK and ELPLLSPPQDK, the amino acid of heavy label are located at peptide section sequence C- respectively On the arginine and lysine of end.
As a preferred embodiment, the concentration of every standard peptide fragment is in the heavy label standard peptide fragment test tube 50fmol。
Embodiment three, oophoroma marker detection.
Step S10 takes heavy label standard peptide fragment test tube, adds in 90-110uL dissolving buffer, concussion thereto Mixing carries out the centrifugation 60s that speed is 13000g using centrifuge, takes supernatant spare；
Step S20 takes 0.1mg haemocyanins peptide hydrolysis to add in 100uL dissolving buffer, shakes mixing, serum peptide fragment is made Solution, it is spare；
Step S30 takes serum peptide fragment solution made of 2uL steps S20, and it is same to add in weight made of 10uL steps S10 thereto In the plain label standard peptide fragment solution in position, it is uniformly mixed, 4uL is taken to carry out chromatography column feed materials；
The setting of chromatographic isolation gradient is as follows：
Time (min) Flow velocity Formic acid concn Acetonitrile ratio Pure water ratio
0 300 0.1% 2% 98%
2 300 0.1% 8% 92%
47 300 0.1% 35% 65%
50 300 0.1% 98% 2%
60 300 0.1% 98% 2%
PRM detection ion table settings are as follows：
M/Z Peptide L/H Protein
574.782753 VEHSDLSFSK light sp|P61769|B2MG_HUMAN
578.789852 VEHSDLSFSK heavy sp|P61769|B2MG_HUMAN
561.81693 VNHVTLSQPK light sp|P61769|B2MG_HUMAN
565.824029 VNHVTLSQPK heavy sp|P61769|B2MG_HUMAN
638.819669 EFQLFSSPHGK light sp|P02787|TRFE_HUMAN
642.826769 EFQLFSSPHGK heavy sp|P02787|TRFE_HUMAN
583.799268 HQTVPQNTGGK light sp|P02787|TRFE_HUMAN
587.806368 HQTVPQNTGGK heavy sp|P02787|TRFE_HUMAN
683.883135 GSPAINVAVHVFR light sp|P02766|TTHY_HUMAN
688.88727 GSPAINVAVHVFR heavy sp|P02766|TTHY_HUMAN
697.814781 AADDTWEPFASGK light sp|P02766|TTHY_HUMAN
701.82188 AADDTWEPFASGK heavy sp|P02766|TTHY_HUMAN
618.347728 DLATVYVDVLK light sp|P02647|APOA1_HUMAN
622.354828 DLATVYVDVLK heavy sp|P02647|APOA1_HUMAN
700.838256 DYVSQFEGSALGK light sp|P02647|APOA1_HUMAN
704.845356 DYVSQFEGSALGK heavy sp|P02647|APOA1_HUMAN
602.324056 LGIWDDFIPK light sp|Q8WXI7|MUC16_HUMAN
606.331156 LGIWDDFIPK heavy sp|Q8WXI7|MUC16_HUMAN
618.845353 ELPLLSPPQDK light sp|Q8WXI7|MUC16_HUMAN
622.852452 ELPLLSPPQDK heavy sp|Q8WXI7|MUC16_HUMAN
Mass spectrograph setting is as follows：
First mass spectrometric, scanning range：500-800m/z, resolution ratio：70000 (@m/z 200), AGC target：3e6, Maximum IT：200ms；After first mass spectrometric 15 two level PRM scannings are triggered according to ingredient lists；
Two level PRM is scanned, Isolation window：2Th, resolution ratio：35000 (@m/z 200), AGC target：3e6, Maximum IT：200ms, Microscans：1, MS2Activation Type：HCD, NCE：27；
Step S40 carries out PRM mass spectrometric datas Skyline analyses, and concentration of the marker in Sample serum in calculating 5.
Each sample carries out 3 repetition PRM detection experiments, and obtained result is as follows：
Protein Name PRM_1(pmol/ml) PRM_2(pmol/ml) PRM_3(pmol/ml)
sp|P61769|B2MG_HUMAN 51.30 62.70 58.9
sp|P02787|TRFE_HUMAN 43.20 39.70 45.8
sp|P02766|TTHY_HUMAN 68.90 72.10 75.4
sp|P02647|APOA1_HUMAN 50.90 55.20 53.7
sp|Q8WXI7|MUC16_HUMAN 71.80 78.40 75.2
Kit provided by the invention and its detection method are than the sensitivity and specificity of the detection method of conventional OVA1 testing cassetes It is relatively high.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Claims (9)

1. a kind of chromatographic column for being applicable in oophoroma PRM detections, including column tube, filler and pipette tips, filler is filled in column In pipe, pipette tips are sleeved on one end of column tube, which is characterized in that the column tube length is 115-125mm, outer diameter 355-365um, Internal diameter is 70-80um；The filler is the C18 reverse phase fillers of 2.5-3.5um grain sizes.
2. the chromatographic column according to claim 1 for being applicable in oophoroma PRM detections, which is characterized in that the column Length of tube is 120mm, outer diameter 360um, internal diameter 75um；The filler is the C18 reverse phase fillers of 3um grain sizes.
3. a kind of oophoroma marker detection kit based on PRM detections, including conventional Mass Spectrometer Method reagent, feature exists In further including heavy label standard peptide fragment test tube, chromatographic column as claimed in claim 1 or 2, dissolving buffer；The heavy label standard peptide fragment test tube includes thyroxine, aPoA -1, β2-microglobulin (Beta2M), ten unique sequence codes in transferrins and cancer antigen 125, wherein each albumen respectively has two sections of peptide section sequences.
4. a kind of oophoroma marker detection kit based on PRM detections according to claim 3, which is characterized in that The heavy label standard peptide fragment be VEHSDLSFSK, VNHVTLSQPK, EFQLFSSPHGK, HQTVPQNTGGK, GSPAINVAVHVFR, AADDTWEPFASGK, DLATVYVDVLK, DYVSQFEGSALGK, LGIWDDFIPK and ELPLLSPPQDK。
5. a kind of oophoroma marker detection kit based on PRM detections according to claim 3 or 4, feature exist In the amino acid of the heavy label of the heavy label standard peptide fragment is located at the essence of peptide section sequence C- ends respectively On propylhomoserin and lysine.
6. a kind of oophoroma marker detection kit based on PRM detections according to claim 3 or 4, feature exist In the concentration of every standard peptide fragment is 50fmol in the heavy label standard peptide fragment test tube.
7. a kind of oophoroma marker detection kit based on PRM detections according to claim 3, which is characterized in that The dissolving buffer is the aqueous solution containing 0.1% formic acid.
8. a kind of detection method of detection kit using as described in claim 3-4 is any, which is characterized in that it include with Lower step：
Step S10 takes heavy label standard peptide fragment test tube, adds in 90-110uL dissolving buffer thereto, shakes mixing, The centrifugation 45-75S that speed is 12000-14000g is carried out using centrifuge, takes supernatant spare；
Step S20 takes 0.08-0.12mg haemocyanins peptide hydrolysis to add in 90-110uL dissolving buffer, shakes mixing, be made Serum peptide fragment solution, it is spare；
Step S30 takes serum peptide fragment solution made of 1.8-2.2uL steps S20, and adds in 9-11uL step S10 systems thereto Into heavy label standard peptide fragment solution in, be uniformly mixed, take 3-5uL carry out chromatography column feed materials；
The setting of chromatographic isolation gradient is as follows：
PRM detection ion table settings are as follows：
M/Z Peptide L/H Protein
574.782753 VEHSDLSFSK light sp|P61769|B2MG_HUMAN
578.789852 VEHSDLSFSK heavy sp|P61769|B2MG_HUMAN
561.81693 VNHVTLSQPK light sp|P61769|B2MG_HUMAN
565.824029 VNHVTLSQPK heavy sp|P61769|B2MG_HUMAN
638.819669 EFQLFSSPHGK light sp|P02787|TRFE_HUMAN
642.826769 EFQLFSSPHGK heavy sp|P02787|TRFE_HUMAN
583.799268 HQTVPQNTGGK light sp|P02787|TRFE_HUMAN
587.806368 HQTVPQNTGGK heavy sp|P02787|TRFE_HUMAN
683.883135 GSPAINVAVHVFR light sp|P02766|TTHY_HUMAN
688.88727 GSPAINVAVHVFR heavy sp|P02766|TTHY_HUMAN
697.814781 AADDTWEPFASGK light sp|P02766|TTHY_HUMAN
701.82188 AADDTWEPFASGK heavy sp|P02766|TTHY_HUMAN
618.347728 DLATVYVDVLK light sp|P02647|APOA1_HUMAN
622.354828 DLATVYVDVLK heavy sp|P02647|APOA1_HUMAN
700.838256 DYVSQFEGSALGK light sp|P02647|APOA1_HUMAN
704.845356 DYVSQFEGSALGK heavy sp|P02647|APOA1_HUMAN
602.324056 LGIWDDFIPK light sp|Q8WXI7|MUC16_HUMAN
606.331156 LGIWDDFIPK heavy sp|Q8WXI7|MUC16_HUMAN
618.845353 ELPLLSPPQDK light sp|Q8WXI7|MUC16_HUMAN
622.852452 ELPLLSPPQDK heavy sp|Q8WXI7|MUC16_HUMAN
Mass spectrograph setting is as follows：
First mass spectrometric, scanning range：500-800m/z, resolution ratio：70000 (@m/z 200), AGC target：3e6, Maximum IT：200ms；After first mass spectrometric 15 two level PRM scannings are triggered according to ingredient lists；
Two level PRM is scanned, Isolation window：2Th, resolution ratio：35000 (@m/z 200), AGC target：3e6, Maximum IT：200ms, Microscans：1, MS2 Activation Type：HCD, NCE：27；
Step S40 carries out PRM mass spectrometric datas Skyline analyses, and calculates concentration of 5 kinds of markers in Sample serum.
9. detection method according to claim 8, which is characterized in that each sample carries out 2-5 and repeats PRM detection examinations It tests.
CN201711396031.3A 2017-12-21 2017-12-21 Oophoroma marker detection kit and detection method based on PRM detections Pending CN108152430A (en)

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CN113376282A (en) * 2021-06-10 2021-09-10 谱天（天津）生物科技有限公司 Kit and detection method for lung cancer protein detection
CN110146635B (en) * 2019-04-28 2022-01-07 北京谷海天目生物医学科技有限公司 Chromatographic analysis column, and kit and device for detecting prosthetic joint infection
CN110133170B (en) * 2019-04-28 2022-01-07 北京谷海天目生物医学科技有限公司 Protein detection method for complex sample
CN115097041A (en) * 2022-06-28 2022-09-23 中国农业科学院北京畜牧兽医研究所 Kit for detecting luteinizing hormone by PRM (platelet-rich plasma) as well as detection method and application

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