CN101271116A - Application of strong anion pre-column in phosphorylation peptide automatization analysis procedure - Google Patents
Application of strong anion pre-column in phosphorylation peptide automatization analysis procedure Download PDFInfo
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Abstract
The invention relates to a liquid-phase chromatogram technology, in particular to a device with easy operation, convenient maintenance and no need of adding additional hardware, and the device can realize the application of the nano-upgrading capillary column high performance liquid-phase chromatogram and a tandem mass spectrum strong anion pre-column in the automatic analysis process of phosphorylated peptide. The invention adopts the strong anion pre-column and a reverse phase C18 separation column as the system for automatic separation and analysis, which successfully solves the problem of the shortcoming of the solid-phase system with the pre-column and the separation column which are both reverse-phase C18, develops the device with easy operation, convenient maintenance and no need of adding additional hardware and realizes the high-efficient and accurate automatic analysis of the phosphorylated peptide sample by the combined usage of the nano-upgrading capillary column high performance liquid-phase chromatogram and the tandem mass spectrum.
Description
Technical field
The present invention relates to liquid chromatography technology, a kind of specifically easy to operate, easy to maintenance, need not to increase the additional hardware device, and can realize receiving upgrading capillary column high performance liquid chromatography and tandem mass spectrum and joined the application of reinforcing yin essence ion pre-column in Phosphorylated Peptide automated analysis process.
Background technology
The modification of posttranslational protein matter is the hot subject of studying in the protein group.Protein phosphorylation is the most common, most important a kind of protein post-translational modification mode, protein phosphorylation and dephosphorylation be the whole process of attemperator's vital movement almost, the propagation that comprises cell is grown and differentiation nervous activity, contraction of muscle, metabolism, tumour generation etc., the main transfer mode of the signal transduction known to protein phosphorylation is still present.
Classic method such as labelled with radioisotope that protein phosphorylation is analyzed, methods such as Edman degraded and thin-layer chromatography.These method complex operations need superb experiment skill and more protein, and have potential Radio Active Hazard.Mass-spectrometric technique has developed into one of important tool of identifying phosphorylated protein.But mass spectrum is identifying that phosphorylated protein still is faced with great challenge now, and it is embodied in: the first, and phosphorylated protein belongs to low-abundance protein in cell; The second, the electronegativity of phosphated peptide section is difficult to it in Mass Spectrometer Method protonated; The 3rd, the mass signal of a large amount of non-phosphorylating peptide section that exists in the enzymolysis product can be flooded the ion signal of Phosphorylated Peptide usually.Therefore, the separation of phosphated peptide section in the complex proteinase hydrolysis products, purifying and enrichment are that mass spectrum is successfully identified an of paramount importance step of phosphorylation.In the research of phosphorylated protein group, use two kinds of different enrichment strategies usually, enriched phosphorus acidifying albumen and enriching phosphated peptide section.The enrichment of phosphorylated protein mainly is to modify the chemical property that changes phosphate group, specific then separation and purification by the antibody of phosphorylating protein and chemical method.That the enrichment use of phosphoeptide section is maximum is immobilization metal affinity chromatography (Immobilized metal affinity chromatography, IMAC (Fe
3+, Ga
3+)).Recent TiO
2And ZrO
2Microtrabeculae, Al (OH)
3, Fe
3O
4/ TiO
2Shell hole nano particle etc. also is used to special purifying and enrichment acid peptide.Another method commonly used is a chemical labeling, specifically phosphate group is modified into the purpose that affine site reaches purifying and enrichment acid peptide by chemical reaction.
Yet, phosphorylating protein and zymolyte sample automatic sampling thereof, compartment analysis remain the huge challenge in the research of phosphorylating protein group. at present the automated analysis of extensive phosphoeptide sample mainly utilize large diameter but short capillary column as pre-column, adopt big flow velocity sample introduction, and then carry out compartment analysis.People such as Licklider (" Automation of nanoscale microcapillary liquid chromatography-tandem massspectromentry with a vented column ", " Analytical chemistry ", P3076-3083,2002) invented a kind of pre-column device of the robotization with escape orifice, can carry out compartment analysis by automatic sampling rapidly and efficiently.And before, the operation of all robotizations all is to adopt the pattern that valve switches to carry out, and whole device makes up very complicated, safeguard inconvenience, and the sample loss amount is quite big.But people's such as Licklider device also exists pre-column to change inconvenient and susceptible to plugging shortcoming.In view of this, people (" Amicrocapillary trap cartridge-microcapillary high-performance liquid chromatographyelectrospray ionization emitter device capable of peptide tandem mass spectrometry atthe attomole level on an ion trap mass spectrometer with automated routine operation " such as Yi in the non-patent literature 2, " Rapid Communication.Mass Spectrometry. ", P2093-2098,2003) further developed this automated system, make that the pre-column stationary phase is easy to change, be convenient to safeguard, and further reduced dead volume, reduced the loss of sample.But, in these systems and since pre-column and separating column all be the anti-phase C18 that adopts as stationary phase, the dead volume between them is bigger to the influence that separates, so still there are defectives such as disengaging time delay, peak broadening and separator window narrow down.And, use the C18 pre-column might cause bigger sample loss because the phosphoeptide water wettability is strong.
Summary of the invention
The object of the present invention is to provide a kind of easy to operate, easy to maintenance, need not to increase the additional hardware device, and can realize receiving upgrading capillary column high performance liquid chromatography and tandem mass spectrum and joined the application of reinforcing yin essence ion pre-column in Phosphorylated Peptide automated analysis process.
For achieving the above object, the technical solution used in the present invention is:
The application of a kind of reinforcing yin essence ion pre-column in Phosphorylated Peptide automated analysis process.
The system of the analysis of being adopted comprises automatic sampler, two six logical automatic transfer valve, miniature three-way valve (3) and miniature four-way valves, automatic sampler links to each other with an end of reinforcing yin essence ion pre-column by the miniature three-way valve, the 4. position of another interface of miniature three-way valve and two six logical automatic transfer valves;
The other end of reinforcing yin essence ion pre-column links to each other with anti-phase C18 separating column by miniature four-way valve, the 3rd interface of four-way valve and the 2. position of two six logical automatic transfer valves, the 4th interface of four-way valve connects the single electrode platinum filament of the required voltage that the electron spray nozzle needle is provided, and the single electrode platinum filament other end links to each other with mass spectrometer;
The other end of anti-phase C18 separating column is an electron spray nozzle needle shape, and it is connected with mass spectrometer;
2. the position of two six logical automatic transfer valves is connected with miniature four-way valve by kapillary a; 4. the position of two six logical automatic transfer valves is connected with the miniature three-way valve by kapillary b; 1. the position and 5. the position be waste liquid outlet, 3. block.
Principle: with reinforcing yin essence ion stationary phase is pre-column, anti-phase stationary phase is a separating column, the Phosphorylated Peptide sample is being enriched under the condition of high pH on the reinforcing yin essence ion pre-column, by changing change pH values or salinity sample is eluted to the reverse phase separation post then, then carry out compartment analysis.Feature of the present invention is.
Beneficial effect of the present invention:
The present invention is a kind of automatic sample handling system that utilizes the pre-column device of reinforcing yin essence ion as the Phosphorylated Peptide sample analysis, promoted to adopt to receive upgrading capillary column high performance liquid chromatography and carry out the development of extensive efficient evaluation Phosphorylated Peptide method with the tandem mass spectrum coupling.The present invention adopts reinforcing yin essence ion-exchange pre-column and the anti-phase C18 separating column system as the robotization compartment analysis, successfully solved the deficiency that pre-column and separating column are anti-phase C18 stationary phase system, adopt reverse phase separation post analyzing proteins zymolyte sample, further reduce the interference of non-phosphorylating sample, and improved the efficient of identifying phosphorylated protein.This system is easy to operate, and is easy to maintenance, need not to increase the device of additional hardware.
Description of drawings
Fig. 1 is one of analytic system device structure of the present invention and operating process synoptic diagram.
Fig. 2 is two of analytic system device structure of the present invention and operating process synoptic diagram.
Fig. 3 is for utilizing analytic system check and analysis mouse orgotein zymolyte figure of the present invention.
Fig. 4 is for utilizing prior art check and analysis mouse orgotein zymolyte figure.
Label is: 2. automatic sampler, 3. miniature three-way, 4. miniature four-way, 5. two six logical automatic transfer valve, 6. mass spectrums, 7. pre-column stationary phase, 8. whole plunger, 9. reinforcing yin essence ion pre-column, 10. anti-phase C18 separating column, 11. the electron spray nozzle needle, 12. single electrode platinum filaments, 13. kapillary b, 14. kapillary a.
Embodiment
Below in conjunction with description of drawings the present invention is done concrete narration.
1. the structure of the pre-column system of reinforcing yin essence ion
Concrete application process is as follows,
1) measures that 10cm is long, internal diameter is the kapillary of 100 μ m, press literature method (Xie, CH etc., " Electrochromatographic evaluation of a silica monolith capillary column forseparation of basic pharmaceuticals ", " Electrophoresis ", P790-797 (2005) makes the whole plunger of 1-2mm.Method is as follows: the 1.06g polyglycol is dissolved in the acetic acid of 10ml 0.01M, slowly drips the 4.5ml tetramethoxy-silicane.Potpourri vigorous stirring in ice bath formed colloidal sol in 40 minutes.Colloidal sol is full of kapillary, is positioned in the column oven after sealing kapillary volume two ends with silicon rubber, 40 ℃ of reactions 24 hours.After question response was finished, the water flushing got final product.Use particle diameter 5 μ m, aperture 200 in the kapillary
The reinforcing yin essence ion stationary phase (Hypersil-5-120 of Thermo company, the Porous 20HQ of Applied Biosystems company) of size is filled the pre-column that is about 2cm;
2) measure that 20cm is long, internal diameter is the kapillary of 75 μ m, the one end is fired the tip into about 5 μ m, uses as the electron spray nozzle needle.Use particle diameter 5 μ m, aperture 100 in the kapillary
The anti-phase C18 stationary phase (the Magic C18AQ of Michrom BioResources company, the ODS-AQ of Daiso company) of size is filled the separating column that is about 12cm.
2. the system of automated analysis: (as shown in Figure 1 and Figure 2) comprises automatic sampler (2), two six logical automatic transfer valve (5), miniature three-way valve (3) and miniature four-way valves (4), automatic sampler (2) links to each other with an end of reinforcing yin essence ion pre-column (9) by miniature three-way valve (3), 4. the position of another interface of miniature three-way valve (3) and two six logical automatic transfer valves (5), the 3rd interface of miniature three-way valve (3) links to each other with automatic sampler (2), and automatic sampler (2) is connected with high performance liquid chromatography (1);
Adopt the reinforcing yin essence ion as pre-column stationary phase (7) in the reinforcing yin essence ion pre-column (9), and the nearly miniature four-way of reinforcing yin essence ion pre-column (9) (4) is held by whole plunger (8) its shutoff.
The other end of reinforcing yin essence ion pre-column (9) links to each other with anti-phase C18 separating column (10) by miniature four-way valve (4), the 3rd interface of miniature four-way valve (4) and the 2. position of two six logical automatic transfer valves (5), the 4th interface of miniature four-way valve (4) connects the single electrode platinum filament (12) of the required voltage that electron spray nozzle needle (11) is provided, and single electrode platinum filament (12) other end links to each other with mass spectrometer (6);
The other end of anti-phase C18 separating column (10) is an electron spray nozzle needle shape (11), and it links to each other with mass spectrometer (6);
2. the position of two six logical automatic transfer valves (5) is connected with miniature four-way valve (4) by kapillary a (14); 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13); 1. the position and 5. the position be waste liquid outlet, 3. block.
Detailed process is as follows: control sample introduction and clastotype by two six logical automatic transfer valves of automatic adjusting.
1) pre-equilibration, 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is led to by kapillary a (14) and miniature four valves (4) and is connect, 2. the position with 3. communicate, make moving phase be full of system, the pre-equilibration automatic sample handling system;
2) go up sample, 4. the position is connected with miniature three-way valve (3) by kapillary b (13), and 4. the position communicates with 3. position, and 2. the position is passed through kapillary a (14) and is connected with miniature four-way valve (4), 2. with 1. communicate, sample uploads to reinforcing yin essence ion pre-column (9);
3) strengthen wash-out, 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. the position with 3. communicate, the sample that uploads to reinforcing yin essence ion pre-column (9) enters anti-phase C18 separating column (10) by wash-out;
4) gradient elution and detection, 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is by kapillary a (14) and miniature four-way valve (4), 2. the position with 3. communicate, sample is gradient elution in anti-phase C18 separating column (10), and eluent enters mass spectrum (6) by the terminal electron spray nozzle needle (11) of anti-phase C18 separating column (10) and detects.
The mouse liver Phosphorylated Peptide sample of the system that adopts automated analysis of the present invention after to enrichment analyzed
1) preparation of mouse orgotein zymolyte: 1mg mouse orgotein, be dissolved in the 20mMTris damping fluid (pH8.1) that contains the 6M guanidine hydrochloride, add DTT (final concentration 5Mm), 37 ℃ of water-baths 2 hours, add iodo-acetamide (final concentration 10mM) then, placed 1 hour the room temperature dark place.With the dilution of 20mM Tris damping fluid, guanidine concentration is reduced to below the 1M again.Press enzyme at last: the mass ratio of albumen (1: 25) adds the pure trypsase of sequence, 37 ℃ of enzyme digestion reactions 20 hours.Adopt freeze-drying behind the anti-phase C18 solid-phase extraction column desalination, mouse orgotein enzymolysis product.
2) ZrO
2Nano material specific enrichment and purifying phosphorylated peptides
(1) mouse orgotein enzymolysis product 50%ACN, 10%HAC (pH=2-3) dissolves as sample solution.
(2) get the ZrO of 100 μ L
2Nano material adds the enzymolysis sample solution of 100 μ L, and balance is 30 minutes under the room temperature, and high speed centrifugation under 35000 * g discards supernatant liquor then.
(3) 50%ACN of adding 400 μ L, 10%HAC, ZrO is cleaned in jolting
2Nano particle 5 minutes, high speed centrifugation under 35000 * g discards supernatant liquor then.
(4) 50%ACN of adding 400 μ L, ZrO is cleaned in jolting
2Nano particle 5 minutes, high speed centrifugation under 335000 * g discards supernatant liquor then.
(5) NH of adding 200 μ L
3.H
2O ultrasonic cleaning ZrO
2Nano particle 5 minutes, high speed centrifugation under 35000 * g is collected supernatant liquor then.
(6) with the NH behind the upper strata wash-out of collecting
3.H
2O puts into freeze drier and concentrates, and freeze-drying is to be analyzed.
3) phosphated peptide section potpourri utilization automatic sampling systematic analysis of the present invention: gradient elution also detects, 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. the position with 3. communicate, sample is gradient elution in anti-phase C18 separating column (10), and eluent enters mass spectrum (6) by the terminal electron spray nozzle needle (11) of anti-phase C18 separating column (10) and detects.
(referring to Fig. 1, Fig. 2) 1) pre-equilibration, 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. the position with 3. communicate, it is 200nL that reverse phase separation capillary column this moment (10) is gone up flow velocity, moving phase consist of 10mM ammonium acetate (pH 7.0), moving phase enters from automatic sampler (2) and makes moving phase be full of system after shunting, pre-equilibration automatic sample handling system 10 minutes;
2) go up sample, 4. the position is connected with miniature three-way by kapillary b (13), 4. the position communicates with 3. position, and 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. with 1. communicate, this moment, flow velocity was 2 μ L/min, moving phase consist of 10mM ammonium acetate (pH 7.0), moving phase all flows out from kapillary a (14) emptying kapillary, after sample enters shunting by automatic sampler (2), the sample of 20 μ L is uploaded on the reinforcing yin essence ion pre-column (9) sample injection time 15 minutes.
3) strengthen wash-out, 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. the position communicates with 3. position, and at this moment flow velocity is 200nL, and moving phase consists of 500mM ammonium acetate (pH2.6), the sample that uploads to reinforcing yin essence ion pre-column (9) enters anti-phase C18 separating column (10), elution time 5 minutes by the wash-out of reinforcing yin essence ion.
4) gradient elution and detection, 4. the position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. the position communicates with 3. position, and at this moment flow velocity is 200nL, and sample is gradient elution in anti-phase C18 separating column (10), gradient is from 0.1% formic acid to 5% acetonitrile/0.1% formic acid, totally 5 minutes; Then from 5% acetonitrile/0.1% formic acid to 35% acetonitrile/0.1 formic acid, totally 60 minutes; Rise to 0.1% formic acid to 80% acetonitrile/0.1 formic acid from 35% acetonitrile/0.1 formic acid again, totally 10 minutes; At last from 80% acetonitrile/0.1 formic acid to 0.1% formic acid, totally 15 minutes.The whole analytical process totally 90 minutes, eluent enters tandem mass spectrum (6) by the terminal electron spray nozzle needle (11) of anti-phase C18 separating column (10) and detects (referring to table 1).
Table 1
(2) result shows that it is 69 minutes (referring to Fig. 3) that reinforcing yin essence ion pre-column piece-rate system is analyzed the chromatographic resolution window of mouse liver Phosphorylated Peptide sample, and the result has 97 phosphated peptide sections and identified.
Comparative example
1) preparation of mouse orgotein zymolyte and ZrO
2The nano material specific enrichment is identical with embodiment 1 with purifying phosphorylated peptides.
2) the phosphated peptide section potpourri uses anti-phase pre-column system automation sample introduction and analysis
Press Yi, EC etc. " A microcapillary trap cartridge-microcapillaryhigh-performance liquid chromatography electrospray ionization emitterdevice capable of peptide tandem mass spectrometry at the attomole levelon an ion trap mass spectrometer with automated routine operation ", " RapidCommunication.Mass Spectrometry. ", P2093-2098 (2003) is changed to anti-phase C18 pre-column (10) with strong cation pre-column (9) among the embodiment 1 and experimentizes.
1) the 4. position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 3. communicate, 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. the position with 1. communicate, this moment, flow velocity was 2 μ L/min, moving phase consist of 0.1% formic acid (pH 3.0), moving phase all flows out from kapillary a (14) emptying post, the sample of 20 μ L uploads on the anti-phase pre-column, sample injection time 15 minutes.
2. the 4. position of two six logical automatic transfer valves (5) is connected with miniature three-way valve (3) by kapillary b (13), 4. the position with 5. communicate, 2. the position is connected with miniature four-way valve (4) by kapillary a (14), 2. the position with 3. communicate, flow velocity is 200nL, adopts linear gradient elution method that sample separation and tandem mass spectrum are detected after shunting.Gradient elution is from 0.1% formic acid to 5% acetonitrile/0.1% formic acid, totally 5 minutes; Then from 5% acetonitrile/0.1% formic acid to 35% acetonitrile/0.1 formic acid, totally 60 minutes; Rise to 0.1% formic acid to 80% acetonitrile/0.1 formic acid from 35% acetonitrile/0.1 formic acid again, totally 10 minutes; At last from 80% acetonitrile/0.1 formic acid to 0.1% formic acid, totally 15 minutes.The whole analytical process totally 90 minutes.
(2) found that the mouse liver Phosphorylated Peptide sample after the enrichment uses anti-phase pre-column piece-rate system that very serious delay separation takes place, its chromatographic resolution window is 50 minutes (referring to Fig. 4), and the result has only 65 phosphated peptide sections to be identified.
The ability of presentation of results reinforcing yin essence ion of the present invention pre-column piece-rate system separating phosphoeptide sample is compared its compartment analysis efficient with the anti-phase pre-column piece-rate system of prior art and obviously is better than anti-phase pre-column sampling system.
Claims (2)
1. the application of reinforcing yin essence ion pre-column in Phosphorylated Peptide automated analysis process.
2. according to the application of the described reinforcing yin essence ion of claim 1 pre-column in Phosphorylated Peptide automated analysis process, it is characterized in that: the system of the analysis of being adopted comprises automatic sampler (2), two six logical automatic transfer valve (5), miniature three-way valve (3) and miniature four-way valves (4), automatic sampler (2) links to each other with an end of reinforcing yin essence ion pre-column (9) by miniature three-way valve (3), the 4. position of another interface of miniature three-way valve (3) and two six logical automatic transfer valves (5); The other end of reinforcing yin essence ion pre-column (9) links to each other with anti-phase C18 separating column (10) by miniature four-way valve (4), the 3rd interface of four-way valve (4) and the 2. position of two six logical automatic transfer valves (5), the 4th interface of four-way valve (4) connects the single electrode platinum filament (12) of the required voltage that electron spray nozzle needle (11) is provided, and single electrode platinum filament (12) other end links to each other with mass spectrometer (6);
The other end of anti-phase C18 separating column (10) is an electron spray nozzle needle shape (11), and it is connected with mass spectrometer (6); 2. the position of two six logical automatic transfer valves is connected with miniature four-way valve by kapillary a (14); 4. the position of two six logical automatic transfer valves is connected with the miniature three-way valve by kapillary b (13); 1. the position and 5. the position be waste liquid outlet, 3. block.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104236984A (en) * | 2013-06-14 | 2014-12-24 | 中国科学院大连化学物理研究所 | Device for purifying biological sample and application thereof |
| WO2022117115A1 (en) * | 2021-06-15 | 2022-06-09 | 广东省农业科学院农业生物基因研究中心 | On-line automatic analysis device and analysis method for phosphoproteomics |
| CN115931482A (en) * | 2023-01-09 | 2023-04-07 | 深圳中国计量科学研究院技术创新研究院 | Multichannel real-time online sampling device and mass spectrometer |
-
2007
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104236984A (en) * | 2013-06-14 | 2014-12-24 | 中国科学院大连化学物理研究所 | Device for purifying biological sample and application thereof |
| WO2022117115A1 (en) * | 2021-06-15 | 2022-06-09 | 广东省农业科学院农业生物基因研究中心 | On-line automatic analysis device and analysis method for phosphoproteomics |
| CN115931482A (en) * | 2023-01-09 | 2023-04-07 | 深圳中国计量科学研究院技术创新研究院 | Multichannel real-time online sampling device and mass spectrometer |
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