CN101412778A - Preparation of polymer material for enriching phosphorylated peptide segments - Google Patents
Preparation of polymer material for enriching phosphorylated peptide segments Download PDFInfo
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- CN101412778A CN101412778A CNA2007101575241A CN200710157524A CN101412778A CN 101412778 A CN101412778 A CN 101412778A CN A2007101575241 A CNA2007101575241 A CN A2007101575241A CN 200710157524 A CN200710157524 A CN 200710157524A CN 101412778 A CN101412778 A CN 101412778A
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- phosphate group
- peptide section
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- zirconium
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Abstract
The invention relates to high-selectivity enrichment of phosphorylated peptide, in particular to a method for preparing polymer materials for enriching phosphorylated peptide fragments. The method adopts monomers provided with phosphate groups to perform polymerization, so as to directly form the polymer materials provided with the phosphate groups. The materials are acted by a solution containing zirconium ions to generate phosphate ester zirconium groups, so that the materials can specially enrich and purify the phosphorylated peptide from a complex proteolysis solution. The method adopts the polymerization method to directly synthesize the polymer materials provided with functional groups, does not need to additionally perform chemical modification, and has the characteristics of simplicity and convenience.
Description
Technical field
The present invention relates to the separation and purification of phosphated peptide section, a kind of specifically preparation method who is used for the polymer materials of enriching phosphated peptide section.
Background technology
The evaluation of decorating site is one of significant challenge in the present protein science research behind the protein translation, and phosphorylation modification is one of most important modification behind the protein translation.Because reversible protein phosphorylation modification reaction is in the signal conduction of cell, proteic active function is being regulated in aspects such as metabolism.Recently, mass-spectrometric technique has developed into one of important instrument in protein phosphorylation qualitative.In the phosphated peptide section mass spectroscopy of phosphorylated protein enzymolysis product, owing to have a large amount of non-phosphorylating peptide sections in the enzymolysis product, the ion signal that these peptide sections produce in ionization process can flood the ion signal of phosphated peptide section, because phosphated peptide section relatively low Ionization Efficiency in mass spectrometric detection, it is detected that these factors have caused phosphated peptide section to be difficult in mass spectrum on the other hand.Separation and enriching phosphated peptide section are the more satisfactory methods of carrying out phosphorylation research at present from the enzymolysis mixture of phosphorylated protein, also are the steps of successfully realizing the mass spectroscopy most critical.In order to improve the detection to phosphated peptide section, affine technology is applied to purifying and enriching phosphated peptide section from the biased sample of complexity.The most frequently used affine technology is an immobilized metal chelating affinity chromatography (IMAC), and phospho-peptide is based on the sequestering action between the chelating ion of itself and stationary phase in the reservation on the IMAC.Metal ion commonly used is iron (Fe
3+) and gallium (Ga
3+), general using iminodiethanoic acid (IDA) is that chelation group is immobilized on chromatographic media with metal ion.Though the IMAC based on iron and gallium is in daily use, its specificity is not strong, thereby some acidity peptides also can keep the detection of disturbing phospho-peptide.
Based on the strong interaction that utilizes between zirconium ion and the phosphate group, we develop the new IMAC method (Zr that based on Zirconium phosphoester recently
4+-IMAC).Compare with conventional IMAC, this method has better specificity to phosphorylated peptide.Be different from general IMAC, at Zr
4+Used chelation group also is a phosphate group among the-IMAC.In use at first with the immobilized Zirconium phosphoester surface that forms on the dielectric surface that phosphate group is modified of zirconium ion, phosphorylated peptide keeps owing to the strong interaction between its phosphate group and the zirconium ion obtains specificity.In order to prepare this chromatographic adsorption material that has phosphate group, need carry out modification to material surface, this needs the multistep chemical reaction, therefore relatively wastes time and energy.The invention provides the method for this material of a kind of more simple preparation.
Summary of the invention
The object of the invention is to provide a kind of preparation method who is used for the sorbing material of enriching phosphated peptide section.The monomer of this method utilization band phosphate group carries out the polymkeric substance that polyreaction forms the band phosphate group, and this polymkeric substance Ao closes Zr
4+After, can be used for the high specific enrichment of phosphorylated peptide.
For achieving the above object, the technical solution used in the present invention is:
A kind of preparation method who is used for the polymer materials of enriching phosphated peptide section, employing has the compound monomer of ethylene linkage and phosphate group, carry out polyreaction, form the phosphate group polymkeric substance, this material through with the zirconium ion effect after generate the Zirconium phosphoester group, can be used for the compound that enriching phosphated peptide section etc. has phosphate group.
The described compound that has ethylene linkage and phosphate group has the structure suc as formula (I),
R is CH
2, CH
2CH
2, CH
2CH
2CH
2One of, phosphate group is present in the monomer with the phosphoric acid ester bond, is phosphoric acid one ester.
Detailed process is,
1) compound that has ethylene linkage and a phosphate group with employing be function monomer, methylene-bisacrylamide as linking agent, add organic pore-creating agent, form reaction mass, carry out the thermal-initiated polymerization reaction, form the phosphate group polymkeric substance;
The mass ratio of function monomer and linking agent is 75-85:55-65, and the add-on of pore-creating agent is 75～85% of a reaction mass volume; Temperature of reaction is 50～80 ℃;
2) the phosphate group polymkeric substance is contacted with 10-100mM zirconium ion solution, generate the Zirconium phosphoester group, the compound that it can be used for enriching phosphated peptide section or has phosphate group.
The present invention has following advantage:
The present invention utilizes the monomer of band bound phosphate groups to carry out the polymkeric substance that polyreaction directly forms the band phosphate group, and its polymer materials can be prepared into the integral post pattern, also can be prepared into graininess.
This method is directly prepared the sorbing material that has phosphate group by the method for polyreaction, need not carry out extra chemical reaction to material, thereby has characteristics simply and easily.
Description of drawings
Fig. 1 is the chemical expression of monomer 2-(methacryloxypropyl) ethyl phosphonic acid ester and linking agent methylene-bisacrylamide polyreaction.
Fig. 2 is enrichment of polymkeric substance sorbing material and the process that detects phospho-peptide.
Fig. 3 be the polyalcohol integral material to the phosphated peptide section in phosphorylated protein-casein enzymolysis liquid enrichment and the MALDI mass spectrum of purifying.
Fig. 4 is that the polyalcohol integral material is to the enrichment of the phosphated peptide section in phosphorylated protein-casein enzymolysis liquid and the MALDI mass spectrum of purifying.
Fig. 5 be the polymer beads material to the phosphated peptide section in phosphorylated protein-casein enzymolysis liquid enrichment and the MALDI mass spectrum of purifying.
Fig. 6 is that the polymer beads material is to the enrichment of the phosphated peptide section in phosphorylated protein-casein enzymolysis liquid and the MALDI mass spectrum of purifying.
Embodiment
The present invention generates the polymkeric substance that has phosphate group by monomer and the linking agent polyreaction of utilizing the band bound phosphate groups, and this polymkeric substance Ao closes Zr
4+After can be used for the enrichment of phosphorylated peptide.In order to prepare polymer materials, at first need the prepared polymer reaction solution.Monomer and linking agent are the major ingredient of reaction solution, form polymer backbone behind their polymerization reaction take places.For the physicochemical property of telomerized polymer, can use multiple monomer, but wherein must comprise a kind of monomer that has phosphate group.Porous material has bigger surface-area, and therefore higher loading capacity is arranged.In order to prepare the porous polymkeric substance, need in reaction solution, add a certain proportion of pore-creating agent.Pore-creating agent is some organic solvents, and they do not participate in polyreaction, in polyreaction their occupied spaces, back takes place and will form the hole.By regulating the ratio of pore-creating agent in the reaction solution size and the physical strength of telomerized polymer mesopore very easily.In addition, generally also need to add a spot of initiator and come initiated polymerization.Acquisition is immersed in Zr with polymkeric substance after having the polymkeric substance of phosphate group
4+Solution (as ZrOCl
2Solution) in, Zr
4+Ion forms the surface that Zirconium phosphoester is modified owing to strong interaction is arranged and immobilized on polymer materials with phosphate group on the polymkeric substance.The polymer materials that Zirconium phosphoester is modified can be used for the phospho-peptide of selective extraction protein enzyme solution.
The polymkeric substance that monomer 2-(methacryloxypropyl) ethyl phosphonic acid ester and linking agent methylene-bisacrylamide polyreaction generate has good wetting ability, and is therefore non-specific very little, is the good carrier of affinity chromatography.Below two embodiment all adopt above-mentioned system, but the present invention is not limited to above-mentioned system.
The preparation of embodiment 1 polyalcohol integral pole
Integral post is a kind of special liquid-phase chromatographic column, and it is polymer reaction liquid to be poured into the method for passing through in-situ polymerization in the column jecket form porous, successive polymkeric substance.Polyalcohol integral pole because a lot of through holes are arranged post press very low because convective mass transfer and resistance to mass transfer is very little is suitable for sample pretreatment very much.Present embodiment has prepared the polyalcohol integral capillary column of band phosphate group and has used it for the enrichment of phospho-peptide.
1) kapillary pre-treatment
, be 7.0 with deionized water rinsing kapillary to flowing liquid pH value at first, with methanol solution flushing capillary column 10min, dry up then with nitrogen with 0.1M NaOH solution flushing kapillary void column 1h.Inject the mixture of methyl alcohol and methacryloxypropyl-Trimethoxy silane in the kapillary.Reacted 5-24 hours to 70 degree temperature at 20 degree.Then with methyl alcohol and water flushing.Dry up stand-by at last with nitrogen.
2) preparation of polyalcohol integral pole
With 2-(methacryloxypropyl) ethyl phosphonic acid esters is function monomer, methylene-bisacrylamide is a linking agent, lauryl alcohol, dimethyl sulfoxide (DMSO) and N, dinethylformamide is a pore-creating agent, monomer, linking agent, pore-creating agent are respectively 13%, 10% and 77% uniform mixing by mass percentage, add initiator consumption be 1% of polymer monomer consumption, behind mixed solution usefulness sonic oscillation 15min, reaction solution is sucked in the pretreated empty kapillary, two ends capillaceous are sealed and are dipped in 60 ℃ of water-baths with the little plug of rubber react 8-24h.After reaction is finished, be moving phase,, remove some low polymerization degree materials that the pore-creating agent in the bed, residual reaction reagent and reaction produce with the about 2h of liquid chromatography pump flushing capillary column with methyl alcohol;
As shown in Figure 1, monomer, initiator and pore-creating agent constitute stable homogeneous reaction system before the reaction beginning, and when temperature was elevated to certain value, decomposition of initiator produced radical polymerization.When reaction proceeded to certain degree, polymer chain settled from solution and forms stable dispersion system, was about the small-particle about 1 μ m through further polymerization, crosslinked sclerosis formation diameter.These particles with the interconnection formation of chemical bond bigger " bunch ", these " bunch " the final accumulation form a complete polymeric system.Because the monomer that is adopted, 2-(methacryloxypropyl) ethyl phosphonic acid esters have a phosphate group, and this group does not react in polymerization process, thereby makes the surface of polymer material of generation have phosphate group.After this material Ao closes zirconium ion, form the Zirconium phosphoester sorbing material that phospho-peptide is had the specific adsorption effect.
3) zirconium ion is immobilized
With 20mM zirconium oxychloride solution flushing kapillary, make phosphate group and zirconium on the polymer column bed form stable title complex, with pH be 2 formic acid solution flushing then, standby.
4) selective enrichment of phospho-peptide
The alpha-casein of the preparation of sample solution: 1mg and beta-casein be dissolved in 1mL respectively, in the Ammonium bicarbonate food grade solution of 50mM (pH 8.2), add trypsinase according to the ratio with tryptic mass ratio 40:1 and carry out enzyme digestion reaction, the reaction times is 16h, and hydrolysis temperature is controlled at 37 ℃.It is standby that the proteolysis solution that obtains places-30 ℃ of refrigerators to preserve.
The enrichment of phosphated peptide section: the pH with formic acid adjusting-casein enzymolysis liquid is 2, get 2pmol and contain the integral post of the enzymolysis solution of 50% acetonitrile by 15cm, successively with the formic acid solution that contains 200mM sodium-chlor pH 2, the formic acid solution of pH 2, the flushing kapillary, ammonia soln with pH 10 washes kapillary at last, the phosphated peptide section of enrichment is eluted in the centrifuge tube, after the lyophilize, detect through substance assistant laser desorpted ionization flight time mass spectrum (MALDI), the spectrogram that obtains is seen Fig. 3, among the figure: [M+H] of sequence number 1
+Be 2061.83, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 2
+Be 2352.85, have 4 phosphorylation sites on its pairing peptide section;
[M+H] of sequence number 3
+Be 2966.16, have 4 phosphorylation sites on its pairing peptide section;
Strong peak in Fig. 3 is the peak of phospho-peptide, and this explanation phospho-peptide has obtained specific enrichment in the integral post that Zirconium phosphoester is modified.With-casein, the spectrogram that is obtained is seen accompanying drawing 4 with integral post enriching of alpha-casein enzymolysis liquid sample,
Among the figure [M+H] of sequence number 1
+Be 1237.5, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 2
+Be 1331.53, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 3
+Be 1411.5, have 2 phosphorylation sites on its pairing peptide section;
[M+H] of sequence number 4
+Be 1466.61, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 5
+Be 1539, have 2 phosphorylation sites on its pairing peptide section;
[M+H] of sequence number 6
+Be 1594.7, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 7
+Be 1660.79, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 8
+Be 1832.83, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 9
+Be 1847.69, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 10
+Be 1927.69, have 2 acidifying sites on its pairing peptide section;
[M+H] of sequence number 11
+Be 1944, have 2 phosphorylation sites on its pairing peptide section;
[M+H] of sequence number 12
+Be 1951.95, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 13
+Be 2079.04, have 1 phosphorylation site on its pairing peptide section;
[M+H] of sequence number 14
+Be 2619.04, have 4 phosphorylation sites on its pairing peptide section;
[M+H] of sequence number 15
+Be 2720.91, have 5 phosphorylation sites on its pairing peptide section;
[M+H] of sequence number 16
+Be 3008.01, have 4 phosphorylation sites on its pairing peptide section;
[M+H] of sequence number 17
+Be 3087.99, have 5 phosphorylation sites on its pairing peptide section;
Clearly handle the back phosphorylated peptide and obtain good enrichment with this integral post.
The material of the present invention's preparation is mainly used in the enrichment acid peptide of high specific, because Zirconium phosphoester and phosphate group have strong interaction, it also can be used for the compound of other phosphoric acid group of enrichment; As shown in Figure 2, it has provided the process of utilizing the enrichment of this sorbing material and detecting phospho-peptide.The phospho-peptide that purifying comes out from single proteinic enzymolysis solution can directly use the MALDI mass spectrograph to analyze, and need utilize LC-MS and database retrieval to identify wherein phosphorylated peptide for the phosphorylated peptide that purifies in complex sample such as the protein group sample.
The preparation of embodiment 2 polymer beads
Most liquid-phase chromatographic columns, solid-phase extraction column all use granulated filler such as agarose particle, organic polymer particles, particles of inorganic material as stationary phase, and present embodiment has prepared the polymer beads of band phosphate group and used it for the enrichment of phospho-peptide.
1) preparation of polymer beads
(proportioning is with embodiment 1) is loaded in the centrifuge tube polymerization liquid, seals and is dipped in 60 ℃ of water-baths and react 8-24h.After reaction is finished, grind to form particle and soak some low polymerization degree materials of removing pore-creating agent, residual reaction reagent and reaction generation with methyl alcohol.
2) zirconium ion is immobilized
The particle of preparation is immersed in 20mM zirconium oxychloride solution, vibrates 15 minutes, and is centrifugal, collects settling, obtains the Zirconium phosphoester polymer beads
3) enrichment of phosphated peptide section
Get 2pmol-casein enzymolysis liquid and put into and contain 0.4mg particulate 100mM sodium-chlor, in the formic acid solution of 50% acetonitrile pH 2, vibration, after centrifugal, abandoning supernatant is with the formic acid solution washing settling of 200mM sodium-chlor pH 2, after centrifugal, abandoning supernatant is with the formic acid solution washing settling of pH 2, after centrifugal, abandoning supernatant is handled settling with the ammonia soln of pH 10 at last, supernatant liquor is collected in centrifugal back, after the lyophilize, use maldi analysis, spectrogram is seen accompanying drawing 5.Use identical method to be used for-enrichment of casein enzymolysis liquid phosphated peptide section, the MALDI spectrogram that is obtained is seen accompanying drawing 6.From spectrogram, significant peak all is the peak of phospho-peptide, illustrates that phospho-peptide has the enrichment of high specific on polymer beads.
Claims (5)
1. preparation method who is used for the polymer materials of enriching phosphated peptide section, it is characterized in that: adopt the compound monomer that has ethylene linkage and phosphate group, carry out polyreaction, form the phosphate group polymkeric substance, this material through with the zirconium ion effect after generate the Zirconium phosphoester group, can be used for the compound that enriching phosphated peptide section etc. has phosphate group.
2. according to the described preparation method of claim 1, it is characterized in that: the described compound that has ethylene linkage and phosphate group has the structure suc as formula (I),
R is CH
2, CH
2CH
2, CH
2CH
2CH
2One of, phosphate group is present in the monomer with the phosphoric acid ester bond, is phosphoric acid one ester.
3. according to the described preparation method of claim 1, it is characterized in that:
Detailed process is,
1) compound that has ethylene linkage and a phosphate group with employing be function monomer, methylene-bisacrylamide as linking agent, add organic pore-creating agent, form reaction mass, carry out the thermal-initiated polymerization reaction, form the phosphate group polymkeric substance;
The mass ratio of function monomer and linking agent is 75-85:55-65, and the add-on of pore-creating agent is 75～85% of a reaction mass volume; Temperature of reaction is 50～80 ℃;
2) the phosphate group polymkeric substance is contacted with 10-100mM zirconium ion solution, generate the Zirconium phosphoester group, the compound that it can be used for enriching phosphated peptide section or has phosphate group.
4. according to the described preparation method of claim 1, it is characterized in that: described pore-creating agent is a lauryl alcohol, dimethyl sulfoxide (DMSO) and N, and dinethylformamide, lauryl alcohol, dimethyl sulfoxide (DMSO) and N, the volume ratio of dinethylformamide is 18-22:25-30:3-7.
5. according to the described preparation method of claim 1, it is characterized in that: initiator is Diisopropyl azodicarboxylate, n-Butyl Lithium or ammonium sulfate or Sulfothiorine, and the add-on of initiator is 0.1～5% of function monomer and a linking agent gross weight.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749884A (en) * | 2016-11-18 | 2017-05-31 | 武汉理工大学 | A kind of phosphorylation peptide gathering material and preparation method and application |
| CN106770867A (en) * | 2016-11-18 | 2017-05-31 | 武汉理工大学 | A kind of method for being enriched with detection phosphorylated protein |
| CN109806778A (en) * | 2019-03-14 | 2019-05-28 | 东华大学 | A kind of fixed silane zirconium interface modification polyvinylidene fluoride film and its preparation and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4830273B2 (en) * | 2004-07-05 | 2011-12-07 | 東ソー株式会社 | Cross-linked copolymer and fluorine ion adsorbent comprising the same |
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2007
- 2007-10-17 CN CN2007101575241A patent/CN101412778B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749884A (en) * | 2016-11-18 | 2017-05-31 | 武汉理工大学 | A kind of phosphorylation peptide gathering material and preparation method and application |
| CN106770867A (en) * | 2016-11-18 | 2017-05-31 | 武汉理工大学 | A kind of method for being enriched with detection phosphorylated protein |
| CN109806778A (en) * | 2019-03-14 | 2019-05-28 | 东华大学 | A kind of fixed silane zirconium interface modification polyvinylidene fluoride film and its preparation and application |
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