CN101196527A - Method for fast enriching and appraising phosphopeptide on MALDI-TOF-MS sample target - Google Patents
Method for fast enriching and appraising phosphopeptide on MALDI-TOF-MS sample target Download PDFInfo
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- CN101196527A CN101196527A CNA2006101621194A CN200610162119A CN101196527A CN 101196527 A CN101196527 A CN 101196527A CN A2006101621194 A CNA2006101621194 A CN A2006101621194A CN 200610162119 A CN200610162119 A CN 200610162119A CN 101196527 A CN101196527 A CN 101196527A
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Abstract
The invention relates to a method of fast enrichment and fast identification on the phosphoeptide of the modificatory sample target by the matrix-assisted laser-desorption/time-of-flight mass spectrometry (MALDI-TOF-MS), which is to physically adsorb the fine magnetic nano particle onto the sample target, wash the sample after the sample is loaded to remove the non phosphoeptide and phosphate, add the matrix to make cocrystallization with the phosphoeptide absorbed on the sample target and then conduct the MALDI-TOF-MS analysis. The method does not relate to the chemical reaction without pre-processing to the target, which can modify the target MALDI made of different substrate materials; A plurality of samples complete their enrichment at the same time on the modified target MALDI; There are no sample transfer in the whole process from the sample loading to the mass spectrometric analysis. The invention is easy, fast and sensitive to operate, which has a good application value in the large-scale analysis of the phosphoproteome.
Description
Technical field
The invention belongs to the analytical chemistry field, is a kind of enrichment, the method for identifying phosphoeptide.Be exactly specifically a kind of on ground substance assistant laser parsings/ionization time of-flight mass spectrometer (MALDI-TOF-MS) the sample target of modifying the method for fast enriching, evaluation phosphoeptide.
Background technology
The phosphorylation modification of protein is one of covalent modification important in the biosome, and life processes such as the propagation of pair cell, differentiation, signal transduction, Apoptosis have important regulation.In recent years, mass spectrum (MS) had been widely used in the evaluation of phosphorylating protein.Because the stoichiometric number of phosphorylating protein is lower in the cell/tissue, and there is signal suppressing in non-phosphorylating peptide section to phosphoeptide during mass spectrophotometry, makes direct mass spectrophotometry phosphorylating protein or phosphoeptide have very big difficulty.Enrichment or separating phosphoeptide optionally from the amounts of protein peptide hydrolysis can promote the Mass Spectrometer Method of phosphoeptide.
At present, the method for enrichment acid peptide the most effective, commonly used is immobilized metal affinity chromatography (IMAC).In IMAC, positively charged metal-chelating ion and electronegative phosphoeptide produce electrostatic attraction, cause phosphoeptide to be retained on the immobilized metal-chelating ion.Some metal oxides and oxyhydroxide such as TiO
2, ZrO
2, Al (OH)
3Deng the enrichment that also is used for phosphoeptide.The step of using IMAC enrichment acid peptide to adopt usually: the sample of phosphoric acid peptide loads on IMAC post or ZipTip
MCOn, clean and remove non-phosphopeptide, the phosphoeptide that keeps is eluted, carry out ESI/MALDI-TOF-MS after the desalination and analyze; The IMAC filler that perhaps will be adsorbed with phosphoeptide is directly transferred to and is carried out MALDI-TOF-MS on the MALDI target and analyze.These operation stepss are very loaded down with trivial details, cause sample loss easily, are not suitable for the analysis of micro-example.Recently, bibliographical information two new methods, i.e. bonding Fe respectively on silicon chip
3+-aminotriacetic acid and basic zirconium phosphate directly load on sample enrichment acid peptide on the silicon chip of modification, carry out MALDI-TOF-MS then and analyze.This enrichment method is very complicated to the chemical modification process of target, can not be used for MALDI-TOF-MS and analyze stainless steel target commonly used, handles a plurality of sample difficulties simultaneously.These drawbacks limit its in extensiveization phosphorylating protein group analysis, use.People such as Chen have synthesized a kind of Fe
3O
4/ TiO
2Nano particle, nano particle in centrifuge tube behind the enrichment acid peptide, clean remove non-phosphopeptide and salt after, utilize magnet to isolate nano particle and transfer to and carry out MALDI-MS on the sample target and analyze [Cheng-Tai Chen, Yu-Chie Chen, Fe
3O
4/ TiO
2Core/Shell nanoparticles asaffinity probes for the analysis of phosphopeptides using TiO
2Surface-assisted laser desorption/ionization mass spectrometry is (based on Fe
3O
4/ TiO
2The TiO of affinity probe
2The surface auxiliary laser is resolved ionization massspectrum and is analyzed phosphoeptide), Anal.Chem, 2005,77:5912-5919].Because manual operations in centrifuge tube, transfer process causes sample loss, once can only handle several samples at most, can't satisfy that protein science is extensive, the requirement of high throughput analysis.
Summary of the invention:
The purpose of this invention is to provide a kind of on the MALDI target fast enriching, identify the method for phosphoeptide.
For realizing the purpose of invention, it may further comprise the steps the technical scheme of employing:
(1) nano particle of the material preparation tool magnetic of use selective adsorption phosphoeptide;
(2) nano particle is pipetted on the sample well of MALDI target, apply magnetic field absorption magnetic nano-particle;
(3) sample is loaded on the above-mentioned sample well, hatch with the absorption phosphoeptide;
(4) clean the MALDI target, remove non-phosphopeptide and salt;
(5) remove magnetic field, add the phosphoeptide cocrystallization of matrix and enrichment;
(6) utilize MALDI-TOF-MS to analyze phosphoeptide.
The principle of this scheme is as follows:
The nano particle that invention is used is a kind of difunctional nano particle, has magnetic and alternative absorption phosphoeptide.Can directly carry out enrichment behind the application of sample, utilize the interaction in itself and magnetic field again, nano particle is adsorbed on the MALDI target, prevent that nano particle runs off when non-phosphopeptide and salt are removed in cleaning phosphoeptide.Remove magnetic field after cleaning is finished, phosphoeptide is retained on the MALDI target, can carry out normal MALDI-TOF-MS and analyze.
Obviously, according to principle of the present invention, the operation that applies magnetic field can be carried out in step (4) " cleaning MALDI target is removed non-phosphopeptide and salt " the preceding any time, for example in the after-applied magnetic field of load sample; Or hatch absorption phosphoeptide after-applied magnetic field; Also can before pipetting magnetic nano-particle, apply magnetic field.
Described nano particle preferably uses the nano particle of dual-core architecture, and its kernel is a magnetic material, as Fe
3O
4Skin is the material of alternative absorption phosphoeptide, as TiO
2Or ZrO
2In the document of Chen, the preparation method of this nano particle is promptly disclosed.This nano particle also can adopt the mode with magnetic material and phosphoeptide sorbing material physical connection to make.
For improving the automation of operation level, nano particle is evenly distributed, preferably adopt liquid form when pipetting, as the preparation nanoparticle suspension, pipette with automatic pipettor.
For ease of operation, the fixed magnetic field of external source is preferably adopted in the magnetic field that applies, as placing a magnet to reach the effect of adsorption of nanoparticles at the back side of MALDI target.
Directly add matrix and carry out MALDI-TOF-MS and analyze being adsorbed with on the target of phosphoeptide, the enrichment program before having avoided elutes the phosphoeptide of absorption earlier and puts the shortcoming that target is analyzed again, has improved the utilization factor of sample.
Technical scheme provided by the invention has the following advantages:
1. operate fast and convenient, applied widely.The present invention is based on the physisorption of nano particle to the MALDI target to the modification of MALDI target.Physisorption does not relate to chemical reaction, can be used for the very stainless steel target of inertia of surface, can be used for the MALDI target of other base material, as gold target, silicon chip etc. yet.Do not need before the absorption MALDI target is carried out any processing, whole modification is very simple, quick, can finish the modification to 192 hole targets in five minutes.
2. cost is low, and it is convenient to transform.The present invention applies a magnetic field to the MALDI target, and this magnetic field is only useful at cleaning step, i.e. the interaction of reinforced nano particle and MALDI target prevents that nano particle runs off when cleaning.Can remove magnetic field after cleaning is finished, carry out normal MALDI-TOF-MS and analyze.
3. be specially adapted to the processing of large sample.The MALDI target that the present invention modifies can a plurality of samples of batch processing.Different samples load on respectively in the different sample wells of MALDI target of modification, hatch entire target to be put into cleaning solution after finishing and clean to remove non-phosphopeptide and salt.Analyze 192 hole targets commonly used for MALDI-TOF-MS, on average each sample enrichment time only was 9 seconds, was higher than the time of present enrichment acid peptide method needs far away.
4. be specially adapted to micro-example and handle, pollute little.The present invention is a direct enrichment acid peptide on the MALDI target of modifying, and can carry out MALDI-TOF-MS after enrichment is finished and analyze.The MS that loads on the MAKDI target to the end from sample analyzes, and whole process does not need sample is shifted, and has greatly reduced sample loss, is fit to handle the sample of trace, as peptide hydrolysis in the glue of two-dimensional gel electrophoresis.
Description of drawings:
Fig. 1 is the process flow diagram of invention technical scheme.(a) be the MALDI target of unmodified; (b) for moving the MALDI target that adds behind the nano particle 1,2 magnetic fields for applying; (c) be illustrated in load sample 3 on the target; (d) after expression is cleaned and is removed non-phosphopeptide and salt, the phosphoeptide 4 of enrichment.
Fig. 2 is the MALDI-TOF-MS figure of alpha-casein matter enzymolysis product.(a) the direct MS of not enrichment of enzymolysis product analyzes; (b) enzymolysis product MS after the MALDI target enrichment of modifying analyzes.
Fig. 3 is the MALDI-TOF-MS figure of β casein protein enzymolysis product.(a) the direct MS of not enrichment of enzymolysis product analyzes; (b) enzymolysis product MS after the MALDI target enrichment of modifying analyzes.
Fig. 4 is the MALDI-TOF-MS figure of five standard protein potpourri enzymolysis products.(a) the direct MS of not enrichment of enzymolysis product analyzes; (b) enzymolysis product MS after the MALDI target enrichment of modifying analyzes.
Fig. 5 is the MALDI-TOF-MS figure of beta-casein enzymolysis product after the MALDI target of modifying enrichment simultaneously of different amounts.(a)1000fmol；(b)100fmol；(c)10fmol；(d)0fmol。
Fig. 6 is equivalent to the alpha-casein matter of 200ng and the MALDI-TOF-MS figure after the interior enzymolysis product enrichment of ovalbumin SDS-PAGE glue.(a) alpha-casein matter (b) ovalbumin.
Fig. 7 is equivalent to the alpha-casein matter of 20ng (inferior picomole quantities) and the MALDI-TOF-MS figure after the interior enzymolysis product enrichment of ovalbumin SDS-PAGE glue.(a) alpha-casein matter (b) ovalbumin.
Embodiment
Get the centrifuge tube of the nano particle of a certain amount of preparation, add 0.15% trifluoroacetic acid solution, ultrasonic 1 minute, obtain the nanoparticle suspension of 0.3mg/mL to 1.5mL.
Pipette 0.8 μ L (240ng) nanoparticle suspension distribution respectively on the sample well of ground substance assistant laser parsing/ionization time of-flight mass spectrometer (4700 proteomics Analyzer) stainless steel target, dry the alpha-casein matter enzymolysis product of back loading 2pmol, at room temperature hatched 10 minutes; The target immersion is filled in the double dish of 40mL20% acetonitrile-0.1% trifluoroacetic acid solution, and gentle the cleaning 15 minutes removed non-phosphopeptide and salt on shaking table, takes out with Millpore water and washes target once, adds DHB matrix (20mg/mL-DHB-%H after drying
3PO
4-50%ACN) cocrystallization; Carrying out MALDI-TOF-MS analyzes.During not enrichment, have a lot of non-phosphopeptides to occur, only detect 3 phosphoeptides, as Fig. 2 a, and after the MALDI target enrichment of modifying, most of non-phosphopeptide is removed, and has detected 9 phosphoeptides, shown in Fig. 2 b.Detected phosphoeptide " * " mark.
Pipette 0.8 μ L nanoparticle suspension distribution respectively on the sample well of ground substance assistant laser parsing/ionization time of-flight mass spectrometer stainless steel target, dry the β casein protein enzymolysis product of back loading 2pmol, enrichment is carried out in the operation that repeats embodiment 2, MALDI-TOF-MS analyzes.During not enrichment, only have 1 phosphoeptide to detect, signal to noise ratio (S/N ratio) is very low, and after the enrichment, most of non-phosphopeptide disappears, and detects two phosphoeptides, becomes two peaks the strongest in the spectrogram, shown in Fig. 3 a-b.
To be equivalent to the 60ng bovine serum albumin(BSA), on the sample well of the MALDI target that the horse of 30ng cardiac muscle red eggs are white, alpha-casein, beta-casein and ovalbumin (three of back are phosphorylating protein) potpourri enzymolysis product load on modification, repeat above-mentioned enriching step, carry out MALDI-TOF-MS and analyze.Before the enrichment, a lot of non-phosphopeptides occur, and only detect 5 phosphoeptides, and after the enrichment, non-phosphopeptide are removed effectively, detects 14 phosphoeptides altogether, shown in Fig. 4 a-b." * " represents the phosphoeptide of alpha-casein, and " # " represents the phosphoeptide of beta-casein, "+" represent the phosphoeptide of ovalbumin.
Embodiment 5
The beta-casein matter enzymolysis product of 1pmol, 100fmol, 10fmol and 0fmol is dripped respectively on four adjacent sample wells of MALDI target of modifying, repeat above-mentioned enrichment experiment after, carry out MALDI-TOF-MS and analyze.To 1pmol and 100fmol beta-casein matter enzymolysis product, detect two phosphoeptides respectively, when sample size is reduced to 10fmol, still there is 1 phosphoeptide to detect, produce 11 times signal to noise ratio (S/N ratio), and blank sample occurs without any the peak, shown in Fig. 5 a-d.
Embodiment 6
With 2 μ g (400ng) bovine serum albumin(BSA)s, the horse of 1 μ g (100ng) cardiac muscle red eggs are white, the potpourri of cromoci, alpha-casein and ovalbumin carries out SDS-PAGE and separates, behind the coomassie brilliant blue staining, alpha-casein and ovalbumin band are scaled off with scalpel, carry out enzymolysis in the glue.Get and be equivalent to carry out enrichment on the MALDI target that 100ng (20ng) original protein enzymolysis product loads to modification respectively, carry out MALDI-TOF-MS then and analyze.After the alpha-casein of 100ng and the enrichment of ovalbumin enzymolysis product, detect 5 and 2 phosphoeptides respectively, shown in Fig. 6 a-b.For alpha-casein and the ovalbumin enzymolysis product of 10ng, detect 2 and 1 phosphoeptide after the enrichment, shown in Fig. 7 a-b.
Claims (9)
- A fast enriching on ground substance assistant laser parsing/ionization time of-flight mass spectrometer sample target, identify and it is characterized in that the method for phosphoeptide may further comprise the steps:(1) nano particle of the material preparation tool magnetic of use selective adsorption phosphoeptide;(2) nano particle is pipetted on the sample well of MALDI target, apply magnetic field absorption magnetic nano-particle;(3) sample is loaded on the above-mentioned sample well, hatch with the absorption phosphoeptide;(4) clean the MALDI target, remove non-phosphopeptide and salt;(5) remove magnetic field, add the phosphoeptide cocrystallization of matrix and enrichment;(6) utilize MALDI-TOF-MS to analyze phosphoeptide.
- 2. the method for enrichment acid peptide according to claim 1 is characterized in that: described nano particle is the nano particle of dual-core architecture, and its kernel is a magnetic material; Skin is the material of alternative absorption phosphoeptide,
- 3. the method for enrichment acid peptide according to claim 2 is characterized in that: the kernel of described nano particle is Fe 3O 4
- 4. the method for enrichment acid peptide according to claim 2 is characterized in that: the skin of described nano particle is TiO 2Or ZrO 2
- 5. the method for enrichment acid peptide according to claim 1 is characterized in that: nano particle pipette the employing liquid form.
- 6. the method for enrichment acid peptide according to claim 5 is characterized in that: the form that pipettes the employing nanoparticle suspension of nano particle.
- 7. the method for enrichment acid peptide according to claim 1 is characterized in that: described MALDI target is stainless steel target, gold target or silicon chip target.
- 8. the method for enrichment acid peptide according to claim 1 is characterized in that: the magnetic field that the MALDI target is applied is exogenous fixed magnetic field.
- 9. the method for enrichment acid peptide according to claim 1 is characterized in that: carry out MALDI-TOF-MS and analyze after directly adding the matrix cocrystallization on the target of absorption phosphoeptide.
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| CN101943688A (en) * | 2009-07-10 | 2011-01-12 | 复旦大学 | Method for enriching glycosylated peptide by utilizing mass spectrum target plate |
| CN101750265B (en) * | 2008-12-17 | 2012-02-01 | 中国科学院大连化学物理研究所 | Time-of-flight mass spectrometer for measuring ratio of nano-particle component elements in real time |
| CN102519779A (en) * | 2011-12-06 | 2012-06-27 | 吉林大学 | Concentration and demineralization purification treatment method of biological samples |
| CN103901093A (en) * | 2014-03-13 | 2014-07-02 | 华东理工大学 | Method for preparing microarray chip between hydrophilic phase and hydrophobic phase and carrying out mass spectrum imaging quantitative analysis by using microarray chip |
| CN105572270A (en) * | 2016-02-04 | 2016-05-11 | 吉林大学 | MALDI steel target for conducting single-step enrichment and desalination on biological sample and preparation method of MALDI steel target |
| CN105973974A (en) * | 2016-07-21 | 2016-09-28 | 浙江大学 | MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) detection method of polythiocarbonate type compound |
| CN107167512A (en) * | 2017-06-29 | 2017-09-15 | 浙江和谱生物科技有限公司 | Disposable target plate for substance assistant laser desorpted ionisation mass spectrometry |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101750265B (en) * | 2008-12-17 | 2012-02-01 | 中国科学院大连化学物理研究所 | Time-of-flight mass spectrometer for measuring ratio of nano-particle component elements in real time |
| CN101943688A (en) * | 2009-07-10 | 2011-01-12 | 复旦大学 | Method for enriching glycosylated peptide by utilizing mass spectrum target plate |
| CN102519779A (en) * | 2011-12-06 | 2012-06-27 | 吉林大学 | Concentration and demineralization purification treatment method of biological samples |
| CN102519779B (en) * | 2011-12-06 | 2013-06-12 | 吉林大学 | Concentration and demineralization purification treatment method of biological samples |
| CN103901093A (en) * | 2014-03-13 | 2014-07-02 | 华东理工大学 | Method for preparing microarray chip between hydrophilic phase and hydrophobic phase and carrying out mass spectrum imaging quantitative analysis by using microarray chip |
| CN105572270A (en) * | 2016-02-04 | 2016-05-11 | 吉林大学 | MALDI steel target for conducting single-step enrichment and desalination on biological sample and preparation method of MALDI steel target |
| CN105572270B (en) * | 2016-02-04 | 2017-12-05 | 吉林大学 | A kind of MALDI steel targets that step enrichment and desalination are carried out for biological sample and preparation method thereof |
| CN105973974A (en) * | 2016-07-21 | 2016-09-28 | 浙江大学 | MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) detection method of polythiocarbonate type compound |
| CN105973974B (en) * | 2016-07-21 | 2019-01-11 | 浙江大学 | A kind of detection method of the MALDI-TOF-MS of polythiocarbonates class compound |
| CN107167512A (en) * | 2017-06-29 | 2017-09-15 | 浙江和谱生物科技有限公司 | Disposable target plate for substance assistant laser desorpted ionisation mass spectrometry |
| CN108181418A (en) * | 2017-12-18 | 2018-06-19 | 中国烟草总公司郑州烟草研究院 | A kind of P in Tobacco acidizing protein group determination method |
| CN111380977A (en) * | 2020-03-16 | 2020-07-07 | 东华理工大学 | Sequential ionization analysis method for rare earth samples |
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