CN103185678B - Trace phosphorylated peptide desalting column - Google Patents
Trace phosphorylated peptide desalting column Download PDFInfo
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- CN103185678B CN103185678B CN201110445490.2A CN201110445490A CN103185678B CN 103185678 B CN103185678 B CN 103185678B CN 201110445490 A CN201110445490 A CN 201110445490A CN 103185678 B CN103185678 B CN 103185678B
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Abstract
The invention relates to a trace phosphorylated peptide desalting column, which takes a reversed-phase C18 and polypyrrole composite as a separation material, makes use of electrostatic interaction between positive charges on polypyrrole nitrogen atoms and electronegative phosphate groups and pi-pi stacking interaction of polypyrrole unsaturated double bonds and aromatic amino acid residues to compensate insufficient of C18 stationary phase single hydrophobic interaction and enhance interactions of separation materials and target molecules. Pyrrole and ammonium persulfate are used as raw materials, processed by ultrasound at room temperature, then washed, filled, and sealed to get the trace phosphopeptide peptide desalting column. The polypyrrole-modified quartz material prepared by the invention is high in separation efficiency, purity and stability. The material can be used in acidic conditions and alkaline conditions, so peptides which cannot be detected by conventional C18 stationary phases can be detected, signal to noise ratio is high, and interference is small. The method is simple, does not require sophisticated equipments, can effectively gather phosphorylated peptides of low abundance, is simple in sample analysis operation, and has no complex sample preparation.
Description
Technical field
The present invention relates to a kind of micro-MALDI-PSD desalination pillar TMTip
pPY-C18, this pillar fills in Micro pipette tip in the mode of connecting (Tandem Polypyrrole-C18 packed micropipette tip, referred to as TMTip fixing to polypyrrole and C18
pPY-C18).This invention is applicable to analyze micro-biological sample as protein phosphorylation site in cell, tissue, is the separation method of a kind of comprehensive electrostatic interaction, the interaction of ∏-∏ accumulative facies, hydrophobic interaction.
Background technology
In cell, a large amount of protein all can be translated rear phosphorylation modification, and multi-signal conduction path is participated in this modification directly, and its protein regulation network is the action target of many medicines.The difficult point of analyzing phosphorylation modification is that the abundance of this modified protein is very low, therefore needs Development of Novel material to carry out the enrichment of MALDI-PSD.Metal oxides such as titanium dioxide is one of separation and concentration MALDI-PSD effect best material so far, but need in this way to adopt and carry out wash-out up to the ammonium phosphate solution of 1M, the salt of high concentration is severe jamming testing sample signal in the time further carrying out mass spectrophotometry, corresponding signal can not be detected even completely.
In existing technology, the ZipTip based on anti-phase C18 is the most effective desalination pillar, is also the desalination technology of current unique use.This technology utilizes hydrophobic interaction to retain polypeptide, and salt can not retain at this pillar, after loading, use containing 0.1wt% trifluoro-ethylene (TFA) aqueous cleaning, and the 50wt% acetonitrile solution wash-out containing 0.1wt%TFA for polypeptide, therefore the object that reaches desalination, eluent can be used for mass spectrophotometry.ZipTip
c18shortcoming be effectively to retain phosphorylation modification polypeptide, or the stronger little peptide of some water wettabilities.Phosphate group electronegative on a polypeptide is more, and its water wettability is stronger, thereby more can not be retained, and is usually washed off by 0.1wt%TFA aqueous solution.The present invention utilize in polypyrrole acid solution on nitrogen-atoms the electrostatic interaction of positively charged and electronegative phosphate group, and on polypyrrole on unsaturated double-bond and polypeptide containing aromatic ring amino acid, such as ∏-∏ accumulative facies interaction of H, F, Y, W, strengthen the reservation to each peptide species.The present invention not only can use under acid condition, also can under alkali condition, use, and therefore makes some undissolved polypeptide under acid condition be detected, and has therefore expanded phosphorylation proteomics research field.
Summary of the invention
The object of the present invention is to provide a kind of micro-MALDI-PSD desalination method.A kind of micro-MALDI-PSD desalination pillar TMTip
pPY-C18.The method is simple, can carry out desalination processing to the polypeptide containing high salt concentration, is suitable for mass spectrophotometry.
Realize technical scheme of the present invention:
A kind of micro-MALDI-PSD desalination pillar, this pillar utilizes anti-phase C18 and polypyrrole compound to do parting material, adopts following method to make, and preparation comprises the following steps:
1), take 0.01 gram of silica wool in centrifuge tube, add 1 ml deionized water and 25 microlitre pyrroles, ultrasonic 10 minutes;
2), be that 20 micrograms/microlitre ammonium persulfate aqueous solution adds step 1 fast by 100 microlitre concentration) in the potpourri of gained, eddy current mixes it fast, more ultrasonic 10 minutes;
3), get step 2) obtain polypyrrole modifying silica wool ethanol and clean, and be stored in ethanolic solution;
4), the polypyrrole modifying silica wool being kept in ethanolic solution is filled in to the micro-ZipTip containing C18 reverse phase separation material
c18transfer pipet, fills out it tightly with pipettor;
5), seal micropipet upper end and get final product.
The preparation method of micro-MALDI-PSD desalination pillar of the present invention, is characterized in that comprising the following steps:
1), take 0.01 gram of silica wool in centrifuge tube, add 1 ml deionized water and 25 microlitre pyrroles, ultrasonic 10 minutes;
2), being 20 micrograms/microlitre by 100 microlitre concentration, ammonium persulfate aqueous solution adds step 1 fast) in the potpourri of gained, eddy current mixes it fast, more ultrasonic 10 minutes;
3), get step 2) obtain polypyrrole modifying silica wool ethanol and clean, and be stored in ethanolic solution;
4), by step 3) the polypyrrole modifying silica wool that is kept in ethanolic solution fills in the micro-ZipTip containing C18 reverse phase separation material
c18transfer pipet top, fills out it tightly with pipettor;
5), seal micropipet upper end and get final product.
The application of micro-MALDI-PSD desalination pillar of the present invention, it is characterized in that, be applied to the mass spectrophotometry of tryptose enzymolysis liquid, its way is, by micro-MALDI-PSD desalination pillar successively with each three times of 80wt% acetonitrile solution, 0.1wt%TFA aqueous cleaning containing 0.1wt%TFA; The tryptose enzymolysis liquid that pH 8 is is first adjusted to acidity with 0.1wt%TFA, makes pH 2～3, or direct loading; Then use 0.1wt%TFA aqueous cleaning twice, finally, with the 50wt% acetonitrile solution wash-out containing 0.1wt%TFA, eluent is for mass spectrophotometry.
Effect of the present invention and advantage:
1. the micro-MALDI-PSD desalination pillar that prepared by the present invention, reaction conditions gentleness, without poisonous and harmful reagent.
2. whole technological process is simple and easy to control, and power consumption is few, and productive rate is high, and cost is low, realistic need of production.
3. compared with the existing technology based on anti-phase hydrophobic interaction, the present invention is taking polypyrrole as parting material, utilize the electrostatic interaction between the positive charge on polypyrrole nitrogen-atoms and electronegative phosphate group under acid condition, and ∏-∏ accumulative facies interaction of polypyrrole unsaturated double-bond and aromatic amino acid residue, the deficiency that has made up the fixing mutually single hydrophobic interaction of conventional C18, has strengthened the interaction of parting material and target molecule.Taking pyrroles simple and easy to get and ammonium persulfate as raw material, at room temperature after ultrasonic assisted reaction, then make required micro-MALDI-PSD desalination pillar through processes such as cleaning, filling, sealings.Not only can be used for acid condition, also can be used for alkali condition, desalination is effective, strong to the reservation of MALDI-PSD, applied range.
4. micro-MALDI-PSD desalination method of the present invention, do not need complicated instrument can prepare corresponding micro-MALDI-PSD desalination pillar, the effectively low abundance MALDI-PSD of enrichment, actual sample analysis operation is simple, without complicated sample pre-treatments, processing ease, background interference is little, and analysis speed is fast.
Below in conjunction with drawings and Examples, further the present invention will be described.
Brief description of the drawings
Fig. 1 is the prepared micro-MALDI-PSD desalination pillar pictorial diagram of embodiment.
Fig. 2 is the mass spectrogram of the prepared micro-MALDI-PSD desalination pillar analytical standard MALDI-PSD under acid condition of embodiment.
Fig. 3 is the mass spectrogram of the prepared micro-MALDI-PSD desalination pillar analytical standard MALDI-PSD under alkali condition of embodiment.
Fig. 4 is the mass spectrogram that the prepared micro-MALDI-PSD desalination pillar of enforcement is analyzed zebra fish-egg phosphorylated protein under acid condition.
Fig. 5 is the mass spectrogram that the prepared micro-MALDI-PSD desalination pillar of embodiment is analyzed zebra fish-egg phosphorylated protein under alkali condition.
Can see from figure, various dissimilar MALDI-PSDs have all obtained good desalination effect, detect that the phosphate group number of modifying on a polypeptide is up to 6, and the phosphorylation modification site coverage rate of standard protein is 100%, mass spectrogram signal to noise ratio (S/N ratio) is high, and background interference is few.
Embodiment
Micro-MALDI-PSD desalination method of the present invention, comprises silica wool surface polypyrrole modifying method, the little column preparation method of micro-desalination, pillar preservation and cleaning method, upper quadrat method, sample washing methods and sample elution process.
Silica wool surface polypyrrole modifying method, comprises the following steps:
1), take 0.01 gram of silica wool in centrifuge tube, add 1 ml deionized water and 25 microlitre pyrroles, ultrasonic 10 minutes;
2), being 20 micrograms/microlitre by 100 microlitre concentration, ammonium persulfate aqueous solution adds step 1 fast) in the potpourri of gained, eddy current mixes it fast, more ultrasonic 10 minutes;
3), get step 2) obtain polypyrrole modifying silica wool ethanol and clean, and be stored in ethanolic solution.
The little column preparation method of trace desalination, comprises the following steps:
1), the polypyrrole modifying silica wool being kept in ethanolic solution is filled in to the micro-ZipTip containing C18 reverse phase separation material
c18transfer pipet, fills out it tightly with pipettor;
2), sealing micropipet upper end, avoid silica wool to be in use sucked out.
Pillar is preserved and cleaning method, comprises the following steps:
1) the micro-desalination pillar, preparing is kept in ethanolic solution;
2), use before with cleaning three times containing the 80wt% acetonitrile solution of 0.1wt%TFA, then with the aqueous cleaning three times that contains 0.1wt%TFA.
Upper quadrat method, sample washing methods and sample elution process, comprise the following steps:
1), tryptose enzymolysis liquid (～pH 8) with 0.1wt%TFA regulate acidity to pH 2～3, then carry out resorption several times with pipettor, guarantee that sample is adsorbed on fixing phase completely;
2), with containing the aqueous cleaning of 0.1wt%TFA three times;
3), with the 50wt% acetonitrile solution wash-out containing 0.1wt%TFA.
Embodiment 1
Preparation and the sample analysis of trace MALDI-PSD desalination pillar.
Preparation process is as follows successively:
1), take 0.01 gram of silica wool in centrifuge tube, add 1 ml deionized water and 25 microlitre pyrroles, ultrasonic 10 minutes;
2), being 20 micrograms/microlitre by 100 microlitre concentration, ammonium persulfate aqueous solution adds step 1 fast) in the potpourri of gained, eddy current mixes it fast, more ultrasonic 10 minutes;
3), get step 2) obtain polypyrrole modifying silica wool ethanol and clean, and be stored in ethanolic solution;
4), before analytic sample, by step 3) products therefrom fills in micro-ZipTip
c18transfer pipet top, then successively with containing each three times of 80wt% acetonitrile solution, the 0.1wt%TFA aqueous cleaning of 0.1wt%TFA.Tryptose enzymolysis liquid (～pH8) is first adjusted to acidity with 0.1wt%TFA, pH 2～3, or direct loading.Then use 0.1wt%TFA aqueous cleaning twice, finally, with the 50% acetonitrile solution wash-out containing 0.1wt%TFA, eluent is for mass spectrophotometry.
Prepared micro-MALDI-PSD desalination pillar pictorial diagram is as Fig. 1.
Embodiment 2
Micro-MALDI-PSD desalination pillar prepared by embodiment 1 is for analyzing zebra fish-egg phosphorylated protein
1, preparation TMTIP
pPY-C18a collection of;
2, the micro-MALDI-PSD desalination pillar of step 1 gained is used to 80wt% acetonitrile solution and the aqueous cleaning containing 0.1wt% trifluoro-ethylene (TFA) successively;
3, use pipettor, by the direct loading of trypsase lysate of zebra fish-egg, or regulate the rear loading of pH 2～3;
4, with containing the aqueous cleaning of 0.1%TFA three times;
5, contain the 50% acetonitrile wash-out of 0.1%TFA;
6, mass spectrophotometry experiment, drips in sample target after eluent is mixed with matrix, puts into mass spectrometer (SYNAPT G2HDMS, WATERS, USA), is 200HZ by the frequency adjustment of Ultra-Violet Laser, and the mass spectrogram of gained as shown in Figures 4 and 5.
Claims (3)
1. a micro-MALDI-PSD desalination pillar, is characterized in that: this pillar utilizes anti-phase C18 and polypyrrole compound to do parting material, adopts following method to make, and preparation comprises the following steps:
1), take 0.01 gram of silica wool in centrifuge tube, add 1 ml deionized water and 25 microlitre pyrroles, ultrasonic 10 minutes;
2), be that 20 micrograms/microlitre ammonium persulfate aqueous solution adds in the potpourri of step 1) gained fast by 100 microlitre concentration, eddy current mixes it fast, more ultrasonic 10 minutes;
3), get step 2) obtain polypyrrole modifying silica wool ethanol and clean, and be stored in ethanolic solution;
4), the polypyrrole modifying silica wool being kept in ethanolic solution is filled in to the micro-ZipTipC18 transfer pipet containing C18 reverse phase separation material, with pipettor, it is filled out tightly;
5), seal micro-ZipTipC18 transfer pipet upper end and get final product.
2. the preparation method of micro-MALDI-PSD desalination pillar claimed in claim 1, is characterized in that comprising the following steps:
1), take 0.01 gram of silica wool in centrifuge tube, add 1 ml deionized water and 25 microlitre pyrroles, ultrasonic 10 minutes;
2), the ammonium persulfate aqueous solution of 100 microlitre concentration 20 micrograms/microlitres is added in the potpourri of step 1) gained fast, eddy current mixes it fast, more ultrasonic 10 minutes;
3), get step 2) obtain polypyrrole modifying silica wool ethanol and clean, and be stored in ethanolic solution;
4), polypyrrole modifying silica wool that step 3) is kept in ethanolic solution fills in the micro-ZipTipC18 transfer pipet top containing C18 reverse phase separation material, it is filled out tightly with pipettor;
5), seal micro-ZipTipC18 transfer pipet upper end and get final product.
3. the application of micro-MALDI-PSD desalination pillar claimed in claim 1, it is characterized in that, be applied to the mass spectrophotometry of tryptose enzymolysis liquid, its way is, by micro-MALDI-PSD desalination pillar successively with each three times of 80wt% acetonitrile solution, 0.1wt%TFA aqueous cleaning containing 0.1wt%TFA; PH is that 8 tryptose enzymolysis liquid is first adjusted to acidity with 0.1wt%TFA, makes pH 2～3, or direct loading; Then use 0.1wt%TFA aqueous cleaning twice, finally, with the 50wt% acetonitrile solution wash-out containing 0.1wt%TFA, eluent is for mass spectrophotometry.
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| US8383412B2 (en) * | 2008-10-30 | 2013-02-26 | University Of Louisville Research Foundation, Inc. | Sensors and switches for detecting hydrogen |
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| WO2008054341A2 (en) * | 2005-06-20 | 2008-05-08 | The Research Foundation Of State University Of New York | Method of bioimaging using nanocrystals of fluorescent dyes |
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