CN102788833A - Kit for detecting low-abundance and low-molecular-weight protein spectrum - Google Patents
Kit for detecting low-abundance and low-molecular-weight protein spectrum Download PDFInfo
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- CN102788833A CN102788833A CN2012102529260A CN201210252926A CN102788833A CN 102788833 A CN102788833 A CN 102788833A CN 2012102529260 A CN2012102529260 A CN 2012102529260A CN 201210252926 A CN201210252926 A CN 201210252926A CN 102788833 A CN102788833 A CN 102788833A
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Abstract
The invention discloses a kit for detecting low-abundance and low-molecular-weight protein spectrums. The kit comprises a kit box; a separator, a protein separation and enrichment material, a diluting buffer solution and a cleaning buffer solution are arranged in the kit body; and the protein separation and enrichment material is disposed in the separator and are porous silicon particles, the channel diameter of the material is 1-50nm, and the channel surface of the material has a chemically modified group which is an anion exchange group, a cation exchange group, a hydrophobic group or a polar group. The kit reaches an effect of one-step separation, enrichment and detection of low-molecular-weight proteins in a serum sample through preparing the porous silicon particles in the invention. The kit disclosed in he invention has the advantages of simplicity, portability, and accurate and fast detection method.
Description
Technical field
The invention belongs to the chemical assay technical field, particularly relate to a kind of kit that is used for the low abundance LMWP spectrum of direct detection of complex sample.
Background technology
In recent years, along with proposition and its application in practical study of colony's proteomics notion, the distance between experimental study and the clinical practice will further shorten.Because it is various clinically illness early diagnosiss and the important indicator of estimating the state of an illness that Protein content that causes because of disease in the human body fluid and kind change; Therefore use modern biotechnology, searching is the focus of current research with the specific proteins variation of disease association.Yet, can be used for biomarker that routine clinical detects lacking very also up to now.In theory, because the molecular weight of protein is bigger, in the body fluid circulation more difficult to get access, and the fragment behind some protein degradations but might enter into body fluid or blood through certain approach.With serum is example, and in recent years, the low-molecular-weight scope in the serum proteins group is quite paid close attention to.And developing rapidly and being widely used of mass-spectrometric technique is again that the research of the low abundance low-molecular-weight scope in the serum proteins group provides effective means.
The existing method that detects to LMWP generally is to use separations technology such as electrophoresis that molecular weight of albumen is sieved then with enrichment material target analytes to be carried out enrichment, be aided with follow-up elution step again, thereby carry out Mass Spectrometer Method.This method step is loaded down with trivial details, and the separation of multistep and wash-out operation possibly lost the low molecular protein of trace, and need quite huge checkout equipment.
At present, the difficult point of the analysis of low abundance low molecular weight protein mainly is: 1) in detachment process, lose easily and by the proteasome degradation of other HMW; 2) in the mass spectrum qualification process, covered by the information of other high molecular weight proteins easily; 3) receive micromolecular disturbing effect mass spectrum such as salt, surfactant evaluation etc. more easily.
Summary of the invention
The objective of the invention is to deficiency, a kind of portable kit is provided, be used to detect low abundance LMWP spectrum to prior art.
For this reason; The technical scheme that the present invention adopts is such: a kind of kit that detects low abundance LMWP spectrum; Comprise box body, be provided with tripping device, Protein Separation enrichment material, dilution buffer liquid, cleaning buffer solution in the box body, the Protein Separation enrichment material is placed in the tripping device; It is characterized in that: described Protein Separation enrichment material is the porous silicon particle; Channel diameter is between 1-50nm, and its surface, duct has the chemical modification group, and described chemical modification group is anion exchange groups or cation exchange group or hydrophobic grouping or polar group.
Described chemical modification group is selected from a kind of in amino, sulfydryl or the epoxy radicals.
Further, described Protein Separation enrichment material obtains through following steps:
A) P type boron-doping silicon chip is fixed in the electrolytic cell, by volume for the ratio of 1:4～6 add ethanol and weight concentration be 40% hydrofluorite as electrolytic solution, be anode with the silicon chip; Platinum electrode is a negative electrode, carries out dc electrolysis etching and demoulding, and setting strength of current is 0 ~ 100mA; Etching time is 0 ~ 15 minute; Porous silicon layer behind the etching demoulding dries up with nitrogen with the clean back of alcohol flushing Ultrasonic Pulverization 5 ~ 20 minutes again, and the aperture of the porous silicon particle of gained is at 5 ~ 20nm;
B) above-mentioned porous silicon particle is carried out ozone Oxidation Treatment 5 ~ 20 minutes, form surperficial silicon oxygen bond;
C) the porous silicon particle after the oxidation was immersed in the mixed solution that volumetric concentration is 1% silylating reagent and organic solvent reaction 1 hour, 110 ℃ of dryings 15 minutes in baking oven again, the porous silicon particle behind the evaporate to dryness is cleaned with alcohol immersion;
D) porous silicon in the required aperture of selection carries out finishing with amino silane reagent, hydrosulphonyl silane reagent or epoxy radicals silicone hydride reagent.
Further, described tripping device is centrifuge tube or microtrabeculae or chip-micro fluidic device.
The present invention obtains required low abundance LMWP through tripping device and Protein Separation enrichment material in use, carries out MALDI-TOF then and detects, and obtains high-quality peptide spectrum.Compared with prior art, the present invention has following beneficial effect:
1, the present invention reaches the effect of LMWP in step separation, enrichment and the detection blood serum sample through preparation porous silicon particle.The pore size of porous silicon can accurately be controlled through the electrochemical etching condition, according to the protein molecular weight size that will screen, the porosity of respective material is selected.Can optionally catch the low-molecular-weight biomarker through surface modification technology, simultaneously that content in the human serum is abundant high molecular weight protein and proteinase exclusion have prevented the degraded of low-abundance protein outside the duct.It is a gordian technique of this parting material superior performance that the preparation condition of porous silicon particle is selected.
2, the aperture of material can arbitrarily be regulated and control according to the size of band evaluating objects thing among the present invention, and utilizes porous silicon surface can carry out number of chemical and modify, and optionally from biological sample to be measured, catches the target protein of a certain kind.Simultaneously, can be through in each check point, modifying different groups, the multidimensional analysis of carrying out a kind of sample detects.
3, the particle after the enrichment carries out need not to carry out extra wash-out desalination step after the surface clean among the present invention, can carry out MALDI-TOF and detect, and obtains high-quality peptide spectrum.Detecting the unnecessary porous silicon particle in back can directly carry out gel electrophoresis analysis, and interested differential protein is carried out further isolation identification.Avoided the loss that the sample multistep is handled and elution process is caused.
4, equipment is simply portable among the present invention, and detection method is accurately quick.Whole mensuration process generally can just all be accomplished in a few minutes, and is simple very rapidly, and catches the specificity height, can not destroy the protein of being measured simultaneously.
Description of drawings
Fig. 1 is a Micro-Column Separation device synoptic diagram among the present invention.
Fig. 2 is a centrifuge tube tripping device synoptic diagram among the present invention.
Fig. 3 is 1.0 mgmL
-1Insulin sample MALDI-TOF-MS testing result figure after treatment.
Fig. 4 directly detects MALDI-TOF-MS figure as a result for adopting the present invention to patient serum sample.
Embodiment
The preparation of embodiment one porous silicon
Get P type boron-doping silicon chip (100) crystal formation, be fixed in the electrolytic cell, add 0.5ml ethanol; The 2-3ml mass concentration be 40% hydrofluorite as electrolytic solution, be anode with the silicon chip, platinum electrode is a negative electrode; Carry out dc electrolysis etching and demoulding, setting strength of current is 10 ~ 80mA, and etching time is 5 minutes; Porous silicon layer behind the etching demoulding dries up with nitrogen with the clean back of alcohol flushing Ultrasonic Pulverization 15 minutes again, and the aperture of the porous silicon particle of gained is at 5 ~ 20nm;
Above-mentioned porous silicon particle is carried out ozone Oxidation Treatment 20 minutes, form surperficial silicon oxygen bond;
Porous silicon particle after the oxidation was immersed in the mixed solution that volumetric concentration is 1% amino silane reagent and alcohol solvent reaction 1 hour, 110 ℃ of dryings 15 minutes in baking oven again, the porous silicon particle behind the evaporate to dryness is cleaned with alcohol immersion;
Selecting the aperture is the porous silicon of 10-12nm, carries out finishing with undecenoic acid.
Embodiment two tripping devices one
Referring to Fig. 1, be the embodiment of a kind of tripping device of the present invention.1 is the porous silicon particle among the figure, and 2 is microtrabeculae, and the porous silicon particle is placed in the microtrabeculae, realizes the separation and concentration to specific protein.
Embodiment three tripping devices two
Referring to Fig. 2, be the embodiment of the another kind of tripping device of the present invention.1 is the porous silicon particle among the figure, and 3 is centrifuge tube, and the porous silicon particle is placed in the centrifuge tube, realizes the separation and concentration to specific protein.
The processing and detecting of four pairs of insulin samples of embodiment
Present embodiment, directly detects the insulin sample of low concentration as enrichment material with the porous silicon particle of modifying through undecenoic acid of 26% porosity, and its concrete steps are following:
1, sample absorption
The insulin sample of present embodiment is the medical human parenteral solution, and regular iletin is diluted to 1.0 mgmL with physiological buffer PBS
-1The back is as solution to be measured.
Absorption: get four centrifuge tubes, respectively label 1,2; 3,4, each adds 50 uL solution to be measured; In centrifuge tube 2, add the not silica flour of etching of 3 mg again; Adding 3 mg porositys are 26% porous silicon particle in centrifuge tube 3, and the porosity that in centrifuge tube 4, adds the modification of 3 mg process undecenoic acid is 26% porous silicon particle, and four centrifuge tubes at room temperature vibrate jointly and hatch 1 h.
Separate: above-mentioned suspension is left standstill 2 min, the porous silicon particles settling bottom the centrifuge tube, with liquid-transfering gun careful remove supernatant.
2, sample detection
Clean: in centrifuge tube, add 100 uL cleaning buffer solution (0.2 mmolL
-1The PB damping fluid), behind the vortex vibration mixing suspension is left standstill 2 min, with liquid-transfering gun careful remove supernatant.In centrifuge tube, add 100 uL deionized waters again, behind the vortex vibration mixing suspension left standstill 2 min, with liquid-transfering gun careful remove supernatant.
Detect: the porous silicon particle after will cleaning is transferred on the MALDI target with liquid-transfering gun, natural drying at room temperature, and (2-cyanic acid-4-hydroxycinnamic acid, α-CHCA) overlay on particle surface, natural drying at room temperature with the organic substrate solution of 1 uL then; The sample for preparing is sent into the MALDI-TOF mass spectrometer to be detected.The Mass Spectrometer Method condition: optical maser wavelength 337 nm, laser pulse frequency 200 Hz, accelerating potential 20 kV, sensing range 0 ~ 10 kD, be 220 ns time delay, linear positive ion mode detects.
Reclaim: detect finish after with careful the transferring to the centrifuge tube of porous silicon particle from the MALDI target, add albumen eluent (containing volume ratio and be 25% acetonitrile and 75% trifluoroacetic acid), 20 min are hatched in vibration, and the albumen that adsorbs on the particle is eluted.Above-mentioned suspension is left standstill 2 min, remove supernatant with liquid-transfering gun, the porous silicon particle that obtains behind the solution evaporation can be reused.
Testing result is referring to Fig. 3, and among the figure, horizontal ordinate is represented the karyoplasmic ratio of mass spectra peak, and ordinate is represented the peak intensity of mass spectra peak.The 16th, sample solution with classic method directly point overlay on to detect on the MALDI target plate and obtain; The 17th, after sample adsorbs through silica flour; The silica flour point overlayed on the target plate to detect obtain, the 18th, sample through the enrichment of porous silicon particle after, porous silicon particle point overlayed on to detect on the target plate obtain; The 19th, sample is through after the porous silicon particle enrichment of embodiment one, porous silicon particle point overlayed on to detect on the target plate obtain.The 19 sample signal peaks that obtain are apparently higher than classic method 16.
The processing and detecting of five pairs of patient serum sample of embodiment
Present embodiment, directly detects and analyzes patient's blood serum sample as enrichment material with the porous silicon particle of modifying through undecenoic acid of 26% porosity, and its concrete steps are following:
1, sample absorption
The blood serum sample of present embodiment obtains from hospital; Medical domain technician's routine operation is adopted in the preparation of blood serum sample; Wherein 5-9 is a rectal cancer patient serum, and 10-14 is non-cancer patient's serum, and the blood serum sample for preparing is stored in-80 ℃ ultra low temperature freezer.
Absorption: will blood serum sample add in the centrifuge tube with getting 50 uL after 10 times of the physiological buffer PBS dilutions, adding 3 mg again is 26% porous silicon particle through the porosity of undecenoic acid modification, the suspension that obtains at room temperature vibrates and hatches 1 h.
Separate: above-mentioned suspension is left standstill 2 min, the porous silicon particles settling bottom the centrifuge tube, with liquid-transfering gun careful remove supernatant.
2, sample detection
Clean: in centrifuge tube, add 100 uL cleaning buffer solution (0.2 mmolL
-1The PB damping fluid), behind the vortex vibration mixing suspension is left standstill 2 min, with liquid-transfering gun careful remove supernatant.In centrifuge tube, add 100 uL deionized waters again, behind the vortex vibration mixing suspension left standstill 2 min, with liquid-transfering gun careful remove supernatant.
Detect: the porous silicon particle after will cleaning is transferred on the MALDI target with liquid-transfering gun, natural drying at room temperature, and (2-cyanic acid-4-hydroxycinnamic acid, α-CHCA) overlay on particle surface, natural drying at room temperature with the organic substrate solution of 1 uL then; The sample for preparing is sent into the MALDI-TOF mass spectrometer to be detected.The Mass Spectrometer Method condition: optical maser wavelength 337 nm, laser pulse frequency 200 Hz, accelerating potential 20 kV, sensing range 0 ~ 10 kD, be 220 ns time delay, linear positive ion mode detects.
Reclaim: detect finish after with careful the transferring to the centrifuge tube of porous silicon particle from the MALDI target, add albumen eluent (containing volume ratio and be 25% acetonitrile and 75% trifluoroacetic acid), 20 min are hatched in vibration, and the albumen that adsorbs on the particle is eluted.Above-mentioned suspension is left standstill 2 min, remove supernatant with liquid-transfering gun, the porous silicon particle that obtains behind the solution evaporation can be reused.
Testing result is referring to Fig. 4.Among the figure, horizontal ordinate is represented the karyoplasmic ratio of mass spectra peak, and ordinate is represented the peak intensity of mass spectra peak.5-9 is the rectal cancer patient blood serum sample, and 10-14 is non-cancer patient's blood serum sample, and 15 is untreated blood serum sample.It is 0.15% registration process that tolerance is carried out in the peak position; And peak intensity is carried out normalization handle, being lower than relative signal intensity at 0.5 peak and being designated as 0, relative signal intensity is higher than 0.5 peak and is designated as 1; To screen select peak and carry out hierarchical cluster analysis with SPSS software; Analysis result explanation this method can directly detect the low-abundance protein in the blood serum sample, and can tentatively normal person and cancer patient be distinguished, and has only No. 8 one examples to be included among the normal person.
Claims (4)
1. one kind is detected the kit that low abundance LMWP is composed; Comprise box body; Be provided with tripping device, Protein Separation enrichment material, dilution buffer liquid, cleaning buffer solution in the box body; The Protein Separation enrichment material is placed in the tripping device, it is characterized in that: described Protein Separation enrichment material is the porous silicon particle, and channel diameter is between 1-50nm; Its surface, duct has the chemical modification group, and described chemical modification group is anion exchange groups or cation exchange group or hydrophobic grouping or polar group.
2. a kind of kit that detects low abundance LMWP spectrum as claimed in claim 1 is characterized in that: said chemical modification group is selected from a kind of in amino, sulfydryl or the epoxy radicals.
3. a kind of kit that detects low abundance LMWP spectrum as claimed in claim 1, it is characterized in that: described Protein Separation enrichment material obtains through following steps:
A) P type boron-doping silicon chip is fixed in the electrolytic cell, by volume for the ratio of 1:4～6 add ethanol and weight concentration be 40% hydrofluorite as electrolytic solution, be anode with the silicon chip; Platinum electrode is a negative electrode, carries out dc electrolysis etching and demoulding, and setting strength of current is 0 ~ 100mA; Etching time is 0 ~ 15 minute; Porous silicon layer behind the etching demoulding dries up with nitrogen with the clean back of alcohol flushing Ultrasonic Pulverization 5 ~ 20 minutes again, and the aperture of the porous silicon particle of gained is at 5 ~ 20nm;
B) above-mentioned porous silicon particle is carried out ozone Oxidation Treatment 5 ~ 20 minutes, form surperficial silicon oxygen bond;
C) the porous silicon particle after the oxidation was immersed in the mixed solution that volumetric concentration is 1% silylating reagent and organic solvent reaction 1 hour, 110 ℃ of dryings 15 minutes in baking oven again, the porous silicon particle behind the evaporate to dryness is cleaned with alcohol immersion;
D) porous silicon in the required aperture of selection carries out finishing with amino silane reagent, hydrosulphonyl silane reagent or epoxy radicals silicone hydride reagent.
4. like each described a kind of kit that detects low abundance LMWP spectrum of claim 1-3, it is characterized in that: described tripping device is centrifuge tube or microtrabeculae or chip-micro fluidic device.
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Cited By (5)
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| CN106324070A (en) * | 2015-07-07 | 2017-01-11 | 上海交通大学 | Membrane protein solid phase enrichment-mass spectrum detection combined system and method |
| CN106979973A (en) * | 2016-01-19 | 2017-07-25 | 南京理工大学 | The analysis method of protein interactome under a kind of intracellular environment |
| CN108387424A (en) * | 2018-02-27 | 2018-08-10 | 杭州汇健科技有限公司 | A kind of preparation method and applications for the pretreated porous silica material of biological sample |
| CN108896682A (en) * | 2018-07-18 | 2018-11-27 | 杭州汇健科技有限公司 | A kind of quick mass spectral analysis of peptide fingerprinting spectrum and spectrogram method of discrimination |
| CN112710755A (en) * | 2020-12-22 | 2021-04-27 | 中国人民解放军军事科学院军事医学研究院 | Novel method for analyzing serum/plasma proteome |
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Effective date of registration: 20190520 Address after: 310051 Room 901, 9 storeys, 3 blocks, 475 Changhe Road, Changhe Street, Binjiang District, Hangzhou City, Zhejiang Province Patentee after: Hangzhou Jian Jian Technology Co., Ltd. Address before: 310058 Chemical Experiment Center of Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang Province Patentee before: Zhejiang University |