CN103884807B - A kind of 18o is in the Protein quantitative analysis platform of wire tag and method of operating thereof - Google Patents
A kind of 18o is in the Protein quantitative analysis platform of wire tag and method of operating thereof Download PDFInfo
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- CN103884807B CN103884807B CN201210554634.2A CN201210554634A CN103884807B CN 103884807 B CN103884807 B CN 103884807B CN 201210554634 A CN201210554634 A CN 201210554634A CN 103884807 B CN103884807 B CN 103884807B
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Abstract
The present invention relates to one
18o is in the Protein quantitative analysis platform of wire tag and method of operating thereof.
18o is the online enzymolysis of a kind of egg collection white matter at the Protein quantitative analysis platform of wire tag,
18o at wire tag, peptide separation, be detected on one system, comprising: syringe pump, sample introduction needle, column oven, enzyme reactor, transfer valve, peptide section trapping column, liquid chromatography pump, peptide separation post and mass detector.First protein sample is loaded in enzyme reactor by syringe pump and hatches, and carries out while enzymolysis
18o marks, after enzymolysis completes
18the peptide section of O mark, after trapping column on-line preconcentration, proceeds to peptide separation post and is further separated, detect finally by mass detector.Advantage of the present invention be by proteolysis,
18o mark, peptide separation are integrated.
Description
Technical field:
The present invention relates to one
18o in the Protein quantitative analysis platform of wire tag and method of operating thereof,
18o is the online enzymolysis of a kind of egg collection white matter at the Protein quantitative analysis platform of wire tag,
18o is at wire tag, and peptide separation, is detected as the system of one.
Background technology:
The main isolation technics of proteomics starting stage protein mixture is two-dimensional gel electrophoresis (2DE), with the second dimension size exclusion electrophoresis, protein mixture is fully separated by the first dimension isoelectric focusing electrophoresis, then the albumen colour developing be separated in above gel is made by coomassie brilliant blue staining or silver dye, corresponding protein content in colored intensity representative sample.Just can obtain the relative content of corresponding albumen in two samples by comparing two samples same position albumen colored intensity in two-dimensional gel electrophoresis, on same gel, the intensity of different protein site also represents roughly different proteins relative abundance in same sample simultaneously.But two-dimensional gel electrophoresis operation steps is many, poor reproducibility, extremely sour pole basic protein, memebrane protein can not be analyzed; In biological sample, protein content has very wide dynamic range (crossing over 7-12 the order of magnitude, as blood serum sample) in addition, most of high-abundance proteins and two-dimensional gel electrophoresis can only develop the color.Along with the development of " shotgun " proteomic techniques, two-dimensional gel electrophoresis quantitatively gradually by various based on liquid chromatography be separated the high flux of Mass Spectrometer Method, high sensitivity quantitation method replace.
Isotope labeling is quantitatively the proteomics quantivative approach be most widely used present stage, and mainly comprise dimethyl mark, 18O marks, isotope labeling (SILAC) in body, iTRAQ mark etc.Wherein 18O marks because labelled reagent is relatively cheap; Can mark all kinds of samples such as body fluid, cell, tissues; Mark while enzymolysis, obtain without the need to introducing the many advantages such as additional markers step and apply more widely.At present, the reason limiting this method further genralrlization mainly contains following 2 points: 1,
18o labeling effciency is lower:
18o mark is mainly divided into single stage method and two-step approach, and single stage method operation is comparatively simple, only sample need be dissolved in
18carry out enzymolysis in the solution of O, but this kind of method labeling effciency is very low; Two-step approach labeling effciency is high relative to single stage method, but complex operation is consuming time, needs first to be existed by sample
16carry out enzymolysis in the solution of O, desalination evaporate to dryness after enzymolysis, the peptide section after evaporate to dryness is dissolved in again
18hatch in the solution of O.2,
18o marks existence and to backcross problem, namely adopts classic method to carry out
18after O mark, because proteinase still exists in solution, once encounter
16the solution of O, on mark
18the peptide section of O can backcross again into
16o(JournalofProteomeResearch2009,8,2140-2143; JournalofProteomeResearch2009,8,2157-2163).
For
18o mark exist the problems referred to above, we construct the online enzymolysis of a kind of egg collection white matter,
18o wire tag, peptide separation, be detected on one analytic system.This system by the on-line analysis platform that will comprise enzyme reactor with
18o marks combination, realize the online enzymolysis of protein,
18o, at the fast quantitative analysis of wire tag, peptide separation and detection, has good application prospect in proteomics research.
Summary of the invention
The object of the present invention is to provide the online enzymolysis of a kind of egg collection white matter,
18o wire tag, peptide separation, be detected on one system.This system can be carried out fast
18o, at wire tag, avoids that the complex operation that traditional off-line labeling method brings is consuming time, labeling effciency is low, there is problems such as backcrossing.In order to realize this object, technical scheme of the present invention is:
A kind of
18o at the Protein quantitative analysis platform of wire tag,
18o is at the structure of the Protein quantitative analysis platform of wire tag: sample introduction needle and syringe pump are in transmission connection, and drives sample introduction needle to sample introduction in enzyme reactor by syringe pump; Enzyme reactor is placed in a column oven, and the sample mouth entrance of enzyme reactor is connected with sample introduction needle, and the sample mouth outlet of enzyme reactor connects the first interface of the first four-way or six-way valve; The material inlet of two liquid chromatography pumps is connected with two mobile phase storage tanks respectively, and the material outlet of two liquid chromatography pumps is connected with the entrance of a mixer respectively, and the outlet of mixer connects the second interface of the first four-way or six-way valve; Peptide section trapping column inlet end is connected with the first interface of threeway, and the second interface of threeway is connected with the 3rd interface of the first four-way or six-way valve, and the 3rd interface of threeway is connected with the first interface of the second four-way or six-way valve; 4th interface of the first four-way or six-way valve connects waste liquid collection vessel; The endpiece of peptide section trapping column is connected with the first interface of four-way, and the second interface of four-way is connected with the second interface of the second four-way or six-way valve, and the 3rd interface and the 4th interface of the second four-way or six-way valve connect waste liquid collection vessel; 3rd interface of four-way connects high pressure and powers up metal needle; 4th interface of four-way is connected with the inlet end of peptide separation post, and the endpiece of peptide separation post is connected with the injection port of mass detector.
Described
18o wire tag Protein quantitative analysis platform by proteolysis parts,
18o is integrated at wire tag, peptide separation parts, detecting device.
Enzyme reactor realizes
18o, at the critical component of wire tag, can carry out the peptide section after enzymolysis efficiently while enzymolysis
18o marks; The material preparing enzyme reactor can be the integral material of organic substrate, the integral material of hybrid inorganic-organic, the particulate material of organic substrate; The kind preparing the immobilized enzyme of enzyme reactor is: serine protease.
Described
18o is in the method for operating of the Protein quantitative analysis platform of wire tag:
Flow velocity is regulated by syringe pump, protein sample injection in sample introduction needle is entered in enzyme reactor, enzyme reactor is placed in column oven, after 45min to 90min is hatched in stop, peptide section after enzymolysis in enzyme reactor is injected in trapping column by the first four-way or six-way valve, switch the first four-way or six-way valve and the second four-way or six-way valve, peptide section sample in trapping column will be brought in peptide separation post by liquid chromatography pump mobile phase and be separated further, analyze finally by mass detector.
18during the online enzymatic labelling of O, sample is dissolved in NH
4hCO
3in solution, its concentration range is 20mM to 100mM;
18during the online enzymatic labelling of O, temperature of reaction can be set as 25 DEG C to 40 DEG C;
18during the online enzymatic labelling of O, the time of enzymatic labelling is 45min to 90min.
1, enzyme reactor is adopted to carry out online enzymolysis to protein.
2, the peptide section after enzymolysis is carried out at enzyme reactor situ
18o enzymatic labelling.
3, after having marked, use
18peptide section after mark is flushed in trapping column by the solution of O, then carries out follow-up mass spectrophotometry.
The invention has the beneficial effects as follows:
1, system integration, automaticity are high.
2, owing to not needing off-line enzymolysis, the analysis throughput of system improves greatly.
3, can Direct Analysis albumen, sample pretreatment step reduces, thus the possibility of sample loss, pollution also will reduce.
4, compared to traditional off-line
18o labeling method,
18the sample size that O needs at wire tag is less, is greatly reducing complex operation degree, can ensure very high labeling effciency while the mark time, and there is not the problem backcrossed.
Accompanying drawing explanation
Fig. 1, analysis platform installation drawing;
Fig. 2,
18o is in the mark situation (with unmarked sample contrast) of wire tag to bovine serum albumin(BSA);
Fig. 3,
18o marks the mark situation comparison diagram to bovine serum albumin(BSA) at wire tag and two-step approach off-line;
Fig. 4,
18o backcrosses at wire tag, and (after mark, the same day and mark are after one week for situation
18o marks situation contrast).
Embodiment
Embodiment 1
As shown in Figure 1, this analytical equipment is made up of syringe pump 1, sample introduction needle 2, column oven 3, enzyme reactor 4, first four-way or six-way valve 5, peptide section trapping column 6, peptide separation post 7, second four-way or six-way valve 8, liquid chromatography pump 9, mass detector 10.The feature of this analytical equipment is proteolysis,
18o marks, peptide separation system integration.Operated as follows: regulate flow velocity by syringe pump 1, protein sample injection in sample introduction needle 2 is entered (enzyme reactor 4 is placed in column oven 3) in enzyme reactor 4, after stop hatches 1 hour, peptide section after enzymolysis in enzyme reactor 4 is injected in trapping column 6 by the first four-way or six-way valve 5, switch the first four-way or six-way valve 5 and the second four-way or six-way valve 8, peptide section sample in trapping column 6 will be brought in peptide separation post 7 by liquid chromatography pump 9 mobile phase and be separated further, analyze finally by mass detector 10.
Embodiment 2
Adopt
18o analyzes bovine serum albumin(BSA) in the method for wire tag.The bovine serum albumin solution of 0.1mg/mL (is dissolved in H
2 16the 50mMNH that O is made into
4hCO
3in) be loaded on enzyme post and hatch one hour, hatch rear use H
2 16the 50mMNH that O is made into
4hCO
3mALDI-TOF is used to analyze sample elution; Equally, the bovine serum albumin solution of 0.1mg/mL (is dissolved in H
2 18the 50mMNH that O is made into
4hCO
3in) be loaded on enzyme post and hatch one hour, hatch rear use H
2 18the 50mMNH that O is made into
4hCO
3mALDI-TOF is used to analyze sample elution.The MALDI-TOF analysis result of two increment product as shown in Figure 2.
Embodiment 3
Adopt respectively
18o analyzes bovine serum albumin(BSA) in the two-step approach of wire tag and bibliographical information.The bovine serum albumin solution of 0.1mg/mL (is dissolved in H
2 18the 50mMNH that O is made into
4hCO
3in) be loaded on enzyme post and hatch one hour, hatch rear use H
2 18the 50mMNH that O is made into
4hCO
3mALDI-TOF is used to analyze sample elution
18the labeling effciency of O; Bovine serum albumin solution to another part of 0.1mg/mL (is dissolved in H
2 16the 50mMNH that O is made into
4hCO
3in) in add pancreatin (pancreatin and albumen quality are than being 1:25) enzymolysis and spend the night, then desalination evaporate to dryness is carried out to the solution after enzymolysis, the sample after evaporate to dryness redissolves in H
2 18the 50mMNH that O is made into
4hCO
3in (wherein containing volume ratio be the acetonitrile of 20%), then add pancreatin (pancreatin and albumen quality are than being 1:25) and hatch 24 hours, finally with MALDI-TOF couple
18the labeling effciency of O is analyzed.Two kinds of methods are to bovine serum albumin(BSA)
18the contrast of O labeling effciency as shown in Figure 3.
Embodiment 4
Investigate
18the online labeling method of O is to the situation that backcrosses of sample after bovine serum albumin white marker.By method shown in embodiment 2, bovine serum albumin(BSA) is carried out online
18after O mark, MALDI-TOF analysis is carried out to sample; 10 times of volume H are added in these increment product
2 16the 50mMNH that O is made into
4hCO
3solution, after placing one week, has carried out MALDI-TOF analysis to sample.Analysis result as shown in Figure 4.
Embodiment 5
Adopt
18single stage method and the two-step approach of the online labeling method of O and bibliographical information contrast.Wherein
18the online labeling method of O is that sample is hatched one hour in enzyme reactor; Single stage method is that sample is directly dissolved in H
2 18off-line enzymolysis is carried out in the solution that O is made into; Two-step approach is that sample is first dissolved in H
2 16carry out enzymolysis in the solution that O is made into, the solution after enzymolysis redissolves in H after desalination evaporate to dryness again
2 18hatch in the solution that O is made into.Concrete comparing result is as shown in table 1.
Table 1,
18o is consuming time at the mark of the off-line method of wire tag and bibliographical information, labeling effciency contrasts
| 18O is at wire tag | Single stage method | Two-step approach | |
| Time | 1hour | About 24hour | Be greater than 48hour |
| Labeling effciency | More than 90% | Lower than 80% | About 90% |
Claims (2)
1. one kind
18o, at the Protein quantitative analysis platform of wire tag, is characterized in that:
18o is at the structure of the Protein quantitative analysis platform of wire tag: sample introduction needle (2) and syringe pump (1) are in transmission connection, and drives sample introduction needle (2) to enzyme reactor (4) interior sample introduction by syringe pump (1); Enzyme reactor (4) is placed in a column oven (3), the sample mouth entrance of enzyme reactor (4) is connected with sample introduction needle (2), and the sample mouth outlet of enzyme reactor (4) connects the first interface of the first four-way or six-way valve (5); The material inlet of two liquid chromatography pumps (9) is connected with two mobile phase storage tanks respectively, the material outlet of two liquid chromatography pumps (9) is connected with the entrance of a mixer respectively, and the outlet of mixer connects the second interface of the first four-way or six-way valve (5); Peptide section trapping column (6) inlet end is connected with the first interface of threeway, second interface of threeway is connected with the 3rd interface of the first four-way or six-way valve (5), and the 3rd interface of threeway is connected with the first interface of the second four-way or six-way valve (8); 4th interface of the first four-way or six-way valve (5) connects waste liquid collection vessel; The endpiece of peptide section trapping column (6) is connected with the first interface of four-way, second interface of four-way is connected with the second interface of the second four-way or six-way valve (8), and the 3rd interface and the 4th interface of the second four-way or six-way valve (8) connect waste liquid collection vessel; 3rd interface of four-way connects high pressure and powers up metal needle; 4th interface of four-way is connected with the inlet end of peptide separation post (7), and the endpiece of peptide separation post (7) is connected with the injection port of mass detector (10);
Described
18o wire tag Protein quantitative analysis platform by proteolysis parts,
18o is integrated at wire tag, peptide separation parts, detecting device;
Enzyme reactor realizes
18o, at the critical component of wire tag, can carry out the peptide section after enzymolysis efficiently while enzymolysis
18o marks; The material preparing enzyme reactor is the particulate material of the integral material of organic substrate or the integral material of hybrid inorganic-organic or organic substrate; The kind preparing the immobilized enzyme of enzyme reactor is: serine protease.
2. one kind as claimed in claim 1
18o, in the method for operating of the Protein quantitative analysis platform of wire tag, is characterized in that:
Method of operating is as follows: regulate flow velocity by syringe pump (1), protein sample injection in sample introduction needle (2) is entered in enzyme reactor (4), enzyme reactor (4) is placed in column oven (3), after 45min to 90min is hatched in stop, peptide section after enzymolysis in enzyme reactor (4) is injected in trapping column (6) by the first four-way or six-way valve (5), switch the first four-way or six-way valve (5) and the second four-way or six-way valve (8), peptide section sample in trapping column (6) will be brought in peptide separation post (7) by liquid chromatography pump (9) mobile phase and be separated further, analyze finally by mass detector (10),
18during the online enzymatic labelling of O, NH
4hCO
3be dissolved in H
2 18o makes NH
4hCO
3solution, protein sample is dissolved in NH
4hCO
3in solution, its concentration range is 20mM to 100mM,
18during the online enzymatic labelling of O, temperature of reaction is set as 25 DEG C to 40 DEG C,
18during the online enzymatic labelling of O, the time of enzymatic labelling is 45min to 90min.
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| CN106483236B (en) * | 2015-08-26 | 2018-07-13 | 中国科学院大连化学物理研究所 | A kind of multiplexed protein matter quantitative approach based on equal weight di-methylation label |
| CN106053629A (en) * | 2016-05-17 | 2016-10-26 | 中国烟草总公司郑州烟草研究院 | Integrated online sample preprocessing device and applications thereof |
| DE102016121512A1 (en) | 2016-11-10 | 2018-05-17 | Dionex Softron Gmbh | System, method and use of liquid chromatography |
| CN108831819A (en) * | 2018-04-20 | 2018-11-16 | 中国药科大学 | A kind of equipment and its application in ortho states-denaturation conversion ions source |
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| 在线微酶反应器-微柱液相色谱-电喷雾质谱用于蛋白质快速分析;袁辉明等;《化学通报》;20091231(第5期);427-432 * |
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