CN103102388A - Method for sequentially enriching multi-phosphopeptide and mono-phosphopeptide - Google Patents
Method for sequentially enriching multi-phosphopeptide and mono-phosphopeptide Download PDFInfo
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Abstract
The invention relates to the field of biochemical analysis, and discloses a method for separating and enriching phosphopeptide. By taking a reversed-phase/strong cation exchange mixed mode material as an enrichment material and optimizing parameters such as the type and concentration of buffer salt, the variety and concentration of organic solvent and the like, the invention realizes the sequential elution and selective enrichment of multi-phosphopeptide and mono-phosphopeptide for the first time. Compared with the conventional metal oxide enrichment method, the method of enriching phosphopeptide through the reversed-phase/strong cation exchange mixed mode material has higher sensitivity, higher selectivity and higher multi-phosphopeptide recovery rate. In addition, the enrichment can be performed in a solid-phase extraction (SPE) mode or a dispersion solid-phase extraction mode; the method is simple to operate, high in flux and favorable in repeatability, and is applicable to selective separation and enrichment of phosphopeptide in a complex system; and in conjunction with a mass spectrogram, the material has wide practical prospects in the fields such as research on post-translational modification proteomics and the like.
Description
Technical field
The present invention relates to the enrichment of phosphorylated peptide, specifically a kind of method that adopts anti-phase/strong cation exchange mixed mode material sequences enrichment multi-phosphopeptide and monophosphate peptide.
Background technology
Reversible protein phosphorylation is being brought into play important regulating and controlling effect in cell activities, so the phosphorylated protein in postgraduate's matter sample is very significant for the regulation mechanism that discloses vital process.Biological mass spectrometry is the powerful tool of resolving the phosphorylated peptide segment structure, but mass spectrum still is being faced with huge challenge aspect phosphorylated protein identifying, is mainly the signal suppressing effect of a large amount of non-phosphorylating peptide sections in the low and mass spectrum of abundance due to phosphorylated peptide.Therefore, it is very necessary before mass spectroscopy detects, phosphorylated peptide being carried out selective enrichment.At present, immobilization metal affinity chromatography (IMAC) and titanium dioxide (TiO
2) be the separation and concentration material of the most frequently used phosphorylated peptide.Wherein IMAC uses at present the most traditional the most extensive enriching method, but has reduced the selectivity of this method with the acid non-phosphorylating peptide that phosphorylated peptide flows out altogether.TiO
2Method is used in the phosphorylated protein group because of its this higher phosphorylated peptide selectivity and is got more and more, but the Tripyrophosphoric acid peptide is difficult to from TiO
2Elute on material.
The mixed mode material is subject to the extensive concern of researcher in recent years, as anti-phase/weak anionic exchange material, hydrophilic/cation exchange material and anti-phase/strong cation exchange mixed mode material.Anti-phase in these materials/strong cation exchange mixed mode material is filled in chromatographic column for separating of enriching phosphated peptide.But there are 2 deficiencies in the method enriching phosphated peptide: can not effectively separate multi-phosphopeptide and monophosphate peptide; During the column chromatography operational cost.
With anti-phase/strong cation exchange material and column solid phase extraction pattern or disperse the organic combination of Solid-Phase Extraction pattern, can realize in complex mixture phosphorylated peptide fast, simple, can repeat, high-throughput ground enrichment.Therefore, utilize the different chromatographic effect mechanism of this material, column Solid-Phase Extraction pattern or disperse the Solid-Phase Extraction pattern fast and the characteristic such as simple, realize the selective enrichment of multi-phosphopeptide and monophosphate peptide can promoting the progress of phosphorylated protein group.
Summary of the invention
The object of the present invention is to provide a kind of method for separating and concentrating with phosphorylated peptide of highly selective, high-throughput, simple, good reproducibility, can carry out the select progressively enrichment to trace multi-phosphopeptide and monophosphate peptide.
Adopt anti-phase/strong cation exchange mixed mode material to contact with the phosphorylated protein zymolyte, then order enrichment multi-phosphopeptide and monophosphate peptide;
Described anti-phase/strong cation exchange mixed mode material is to be formed through copolyreaction at Silica Surface by two or more silane reagent, its structural formula is as follows:
Wherein, Silica Gel is silica gel, and NP is that carbonatoms is 4～18 normal chain alkyl, and P is polar group, comprises chlorine atom, bromine atoms, cyano group, Phenylsulfonic acid base or sulfonic group, carboxyl that the normal chain alkyl take carbonatoms as 1～8 is connected.
Enrichment material is anti-phase/strong cation exchange mixed mode material, and the particle diameter of material is 2-0 μ m, and the aperture is 50-
Concrete operations are adopted the column solid phase extraction pattern or are disperseed the Solid-Phase Extraction pattern;
Adopt column solid phase extraction (SPE) pattern with anti-phase/during strong cation exchange mixed mode material sequences enrichment acid peptide, with on the phosphorylated protein zymolyte to the SPE post, adopt the eluting solvent stepwise gradient, be taken up in order of priority enrichment to go out multi-phosphopeptide and monophosphate peptide;
Or adopt to disperse the Solid-Phase Extraction pattern with anti-phase/during strong cation exchange mixed mode material sequences enrichment acid peptide, to directly mix with enrichment material on the phosphorylated protein zymolyte, adopt eluting solvent, use centrifugation, stepwise gradient elution requirement, be taken up in order of priority enrichment to go out multi-phosphopeptide and monophosphate peptide.
Applied sample amount is that enrichment material and protein zymolyte mass ratio are 10-200: 1, and the enrichment temperature is 15-60 ℃.
The present invention has following advantage:
1. the present invention preparation anti-phase/strong cation exchange mixed mode material has shown the characteristics such as highly selective and high-throughput, can realize that multi-phosphopeptide and the effective of monophosphate peptide separate and enrichment when separating and enriching phosphated peptide;
2. the present invention preparation anti-phase/strong cation exchange mixed mode material both can be packed into different lengths easily, and the pillar of different inner diameters can directly add and centrifuge tube again, and is simple to operate, is easy to repetition.Be particularly suitable for the separation and concentration of phosphated peptide section in micro-biological sample;
3. the phosphorylated peptide that obtains of enrichment of the present invention can be directly used in electron spray(ES)-mass spectroscopy (ESI-MS) or substance assistant laser desorpted ionized-flight time mass spectrum (MALD-TOF MS), has improved mass spectral:mass spectrographic detectability and sensitivity.
Description of drawings
Fig. 1 is to the ESI-MS spectrogram after phosphorylated peptide process different methods enrichment in standard phosphorylated protein α-casein (80pmol) enzymolysis product under extraction pattern pattern.(a) α-casein enzymolysis product is through TiO
2Phosphorylated peptide cut after enrichment; (b) the monophosphate peptide fraction of α-casein enzymolysis product after anti-phase/strong cation exchange mixed mode material enrichment; (c) the multi-phosphopeptide cut of α-casein enzymolysis product after anti-phase/strong cation exchange mixed mode material enrichment; (d) mass spectrum of α-after the casein enzymolysis product directly desalts.
Fig. 2 is anti-phase/strong cation exchange mixed mode material MALDI-TOF mass spectrum to phosphorylation peptide gathering front and back in standard phosphorylated protein α-casein (80pmol) enzymolysis product under centrifugal pattern.(a) the multi-phosphopeptide cut of α-casein enzymolysis product after anti-phase/strong cation exchange mixed mode material enrichment; (b) the monophosphate peptide fraction of α-casein enzymolysis product after anti-phase/strong cation exchange mixed mode material enrichment; (c) the non-phosphorylating peptide fraction of α-casein enzymolysis product after anti-phase/strong cation exchange mixed mode material enrichment.
Embodiment
A kind of anti-phase/strong cation exchange mixed mode material (Guo Zhimou; Beam figure; Jin Gaowa; Liang Xinmiao is based on chromatographic separation material and the preparation thereof of Silica Surface copolyreaction, CN200910012845.1,2009), be to be formed through copolyreaction at Silica Surface by two or more silane reagent, its structural formula is as follows:
Wherein, Silica Gel is silica gel, and NP is that carbonatoms is 4～18 normal chain alkyl, and P is polar group, comprises chlorine atom, bromine atoms, cyano group, Phenylsulfonic acid base or sulfonic group, carboxyl that the normal chain alkyl take carbonatoms as 1～8 is connected.
Its preparation process is:
1) to add concentration be in 1%～38% hydrochloric acid or salpeter solution to silica gel, and reflux stirred 1～48 hour, filtered, and was washed to neutrality, was dried to constant weight under 100～160 ℃.
2) to be placed in humidity be 20%～80% atmosphere 24～72 hours to step (1) gained dry silica gel, makes silica gel suction weightening finish 0.5%～10%.
3) Silica Surface copolymerization: step (2) gained hydrated silica gel is placed in glass or tetrafluoroethylene reaction vessel, add organic solvent under nitrogen atmosphere, stir, drip nonpolar silane reagent and polar silanes reagent mixture, keeping temperature is to stir 2～48 hours under 20～200 ℃ of conditions.
Following examples of the present invention are used, and a kind of anti-phase/strong cation exchange mixed mode material structure formula is:
The preparation method: (particle diameter is 40 μ m, and the aperture is 80nm, specific surface area 400m to take the 80g spherical silica gel
2/ g), be placed in the 2000mL glass flask, adding 1200mL concentration is 10% hydrochloric acid soln, reflux 24 hours is cooled to room temperature, filters, and is washed to neutrality, 150 ℃ of dryings 12 hours.Dried silica gel is placed in three mouthfuls of vials of 1500mL, passed into continuously humidity and be 50% nitrogen 48 hours, obtain hydrated silica gel 84g.Under the condition that passes into dry nitrogen, the toluene that adds the 1000mL drying in the hydrated silica gel, stir, then drip 200mmol (80.0mL) octadecyl trichlorosilane alkane and 20mmol (12mL) p-sulfonic acid base styroyl trichlorosilane mixture, under 60 ℃ of room temperatures, stirring reaction is 24 hours.Reaction system is filtered, and uses successively toluene, methylene dichloride, and methyl alcohol, water, tetrahydrofuran (THF), methanol wash, product drying under 80 ℃ of conditions namely got stationary phase shown in structural formula in 12 hours.
The present invention can carry out under Solid-Phase Extraction (SPE) pattern or centrifugal pattern, has the advantages such as simple to operate, flux height and good reproducibility.The invention will be further described by specific embodiment below in conjunction with accompanying drawing, but the present invention is not subjected to the restriction of these embodiment.
The alpha-casein of the preparation of sample solution: 1mg is dissolved in (50mM in the 1mL ammonium bicarbonate soln, pH 8.0), ratio according to the mass ratio 1: 30 (w/w) of trypsinase and alpha-casein adds trypsinase to carry out enzymolysis, 37 ℃ were reacted 12 hours, and got protein enzymatic hydrolyzate and carry out following experimental implementation.
Embodiment 1
With 1mg anti-phase/strong cation exchange mixed mode material packs in the gel suction nozzle, after 2 μ L (80pmol) protein enzymatic hydrolyzate loadings, uses respectively volumetric concentration 30% acetonitrile of 30 μ L/twice of 0.1% formic acid (pH 3) wash-out; Then contain volumetric concentration 50% acetonitrile of the ammonium formiate of 5mM/twice of 0.1% formic acid (pH 3) eluant solution with 30 μ L; Contain volumetric concentration 80% acetonitrile of the ammonium formiate of 20mM/twice of 0.1% formic acid solution (pH 3) wash-out with 30 μ L at last.Elutriant is directly analyzed on mass spectrum.
By Fig. 1 b, 1c and 1d as seen, multi-phosphopeptide in the alpha-casein enzymolysis product and monophosphate peptide can be eluted from anti-phase/strong cation exchange mixed mode material by order, but not phosphorylated peptide by last wash-out out, illustrates that anti-phase/strong cation exchange mixed mode material can specific enrichment and purifying phosphorylated peptides.
The operator scheme of adjusting enrichment is centrifugal, with 1mg anti-phase/strong cation exchange mixed mode material packs in centrifuge tube, 2 μ L (80pmol) alpha-casein enzymolysis solutions are dissolved in the 30%CH of 30 μ L
3CN/0.1% formic acid (pH 3) and and material mixing, hatching 5min, centrifugal rear collection supernatant liquor, precipitation is used 30%CH again
3CN/0.1% formic acid (pH 3) hatching 5min, centrifugal rear merging supernatant liquor.Precipitation and 30 μ L contain the 50%CH of the ammonium formiate of 5mM
3CN/0.1% formic acid solution (pH 3) hatching 5min, centrifugal rear collection supernatant liquor repeats this hatching and centrifugation step, centrifugal rear merging supernatant liquor; Precipitation is hatched 5min, centrifugal rear collection supernatant liquor with the 80%CH3CN/0.1% formic acid (pH3) that 30 μ L contain the ammonium formiate of 20mM at last.Each supernatant liquor is directly analyzed at MALDI-TOF.
By Fig. 2 a, 2b and 2c as seen, the multi-phosphopeptide in the alpha-casein enzymolysis product is not inverted/and strong cation exchange mixed mode material keeps, and directly out (Fig. 2 is a) by wash-out; The monophosphate peptide can be inverted/and strong cation exchange mixed mode material keeps, and can elute (Fig. 2 b) at the salt of low concentration; But not the effect of phosphorylated peptide and material is the strongest at last by wash-out out (Fig. 2 c), illustrates that anti-phase/strong cation exchange mixed mode material can specific enrichment and purifying phosphorylated peptides.
Embodiment 3-6
The weight of adjusting enrichment material is 2mg, 3mg, 4mg, 5mg, other conditions are with embodiment 1, the phosphorylated peptide that obtains after enrichment carries out mass spectroscopy, and experimental result shows that the material of 1mg under extraction pattern operator scheme can keep the phosphorylated peptide in alpha-casein with enrichment 80pmol effectively.
Embodiment 7-9
The applied sample amount of adjusting the alpha-casein enzymolysis solution is 20pmol, 40pmol and 160pmol, other conditions are with embodiment 1, the phosphorylated peptide that obtains after enrichment carries out mass spectroscopy, and experimental result shows that the material of 1mg under extraction pattern operator scheme can keep the phosphorylated peptide in alpha-casein with enrichment 80pmol effectively.
Embodiment 10-12
The pH that adjusts elute soln is 3,4 and 5, and other conditions are carried out selective enrichment and mass spectroscopy with embodiment 1.
Embodiment 13-16
The weight of adjusting enrichment material is 2mg, 3mg, 4mg, 5mg, other conditions are with embodiment 2, the phosphorylated peptide that obtains after enrichment carries out mass spectroscopy, and experimental result shows that the material of 1mg under the centrifugally operated pattern can keep the phosphorylated peptide in alpha-casein with enrichment 80pmol effectively.
Embodiment 17-19
The applied sample amount of adjusting the alpha-casein enzymolysis solution is 20pmol, 40pmol and 160pmol, other conditions are with embodiment 2, the phosphorylated peptide that obtains after enrichment carries out mass spectroscopy, and experimental result shows that the material of 1mg under the centrifugally operated pattern can keep the phosphorylated peptide in alpha-casein with enrichment 80pmol effectively.
Embodiment 20-22
The pH that adjusts elute soln is 3,4 and 5, and other conditions are carried out selective enrichment and mass spectroscopy with embodiment 2.
The present invention is anti-phase/and strong cation exchange mixed mode material has good selective enrichment performance for multi-phosphopeptide and monophosphate peptide, compare with the metal oxide of routine, anti-phase/strong cation exchange mixed mode material enriching phosphated peptide has more highly selective, the higher multi-phosphopeptide rate of recovery and better repeated.Utilize anti-phase/strong cation exchange mixed mode material for the efficient specific adsorption ability of phosphorylated peptide, can be applied to the selective separation enrichment of phosphorylated peptide in complex system, in conjunction with mass spectrum, this material has broad application prospects in fields such as posttranslational modification proteomics researches.
Phosphorylated peptide after the trypsin digestion alpha-casein that table 1ESI-MS and MALDI-TOF detect
Claims (8)
1. the method for an order enrichment multi-phosphopeptide and monophosphate peptide is characterized in that:
Adopt anti-phase/strong cation exchange mixed mode material to contact with the phosphorylated protein zymolyte, then order enrichment multi-phosphopeptide and monophosphate peptide;
Described anti-phase/strong cation exchange mixed mode material is to be formed through copolyreaction at Silica Surface by two or more silane reagent, its structural formula is as follows:
Wherein, Silica Gel is silica gel; NP is that carbonatoms is 4～18 normal chain alkyl; P is the polar group that contains negative ion, comprises chlorine atom, bromine atoms, cyano group, Phenylsulfonic acid base, sulfonic group or carboxyl that the normal chain alkyl take carbonatoms as 1～8 is connected.
2. according to the described method of claim 1, it is characterized in that: enrichment material is anti-phase/strong cation exchange mixed mode material, and the particle diameter of material is 2-50 μ m, and the aperture is 50-
Described two or more silane reagent is nonpolar silane reagent and/or polar silanes reagent.
3. according to the described method of claim 1, it is characterized in that:
Concrete operations are adopted the column solid phase extraction pattern or are disperseed the Solid-Phase Extraction pattern;
Adopt under column solid phase extraction (SPE) pattern anti-phase/during strong cation exchange mixed mode material sequences enrichment acid peptide, with on the phosphorylated protein zymolyte to take anti-phase/strong cation exchange mixed mode material on the SPE post of filler, adopt the eluting solvent gradient, be taken up in order of priority enrichment to go out multi-phosphopeptide and monophosphate peptide;
Or adopt under disperseing the Solid-Phase Extraction pattern anti-phase/during strong cation exchange mixed mode material sequences enrichment acid peptide, to directly mix with enrichment material on the phosphorylated protein zymolyte, adopt eluting solvent, use centrifugation, stepwise gradient elution requirement, be taken up in order of priority enrichment to go out multi-phosphopeptide and monophosphate peptide.
4. according to the described method of claim 3, it is characterized in that: the applied sample amount of sample is that anti-phase/strong cation exchange mixed mode material and phosphorylated protein zymolyte mass ratio are 10-200: 1, and the enrichment temperature is 15-60 ℃; Phosphorylated protein is alpha-casein, beta-casein or ovalbumin.
5. according to the described phosphorylated peptide method for separating and concentrating of claim 3, it is characterized in that:
The eluting solvent that adopts during the enrichment multi-phosphopeptide is that volumetric concentration is acetonitrile, methyl alcohol or the ethanolic soln of 10-50%, pH 2-6; The consumption of elute soln is the multi-phosphopeptide elute soln of 3-40 times of column volume.
6. according to the described phosphorylated peptide method for separating and concentrating of claim 3, it is characterized in that:
The eluting solvent that adopts during wash-out monophosphate peptide is one of following solvents,
1) volumetric concentration that contains the 2-50mM ammonium formiate is 10-50% acetonitrile, methyl alcohol or ethanolic soln, pH 2.2-4.3;
2) volumetric concentration that contains the 2-50mM ammonium acetate is 10-50% acetonitrile, methyl alcohol or ethanolic soln, pH 4.3-5.0;
3) volumetric concentration that contains the 2-50mM potassium primary phosphate is 10-50% acetonitrile, methyl alcohol or ethanolic soln, pH 2.2-4.3;
The consumption of elute soln is the monophosphate peptide elute soln of 3-40 times of column volume.
7. according to the described phosphorylated peptide method for separating and concentrating of claim 3, it is characterized in that:
Adopt and disperse the Solid-Phase Extraction pattern,
1) the phosphorylated protein zymolyte directly with enrichment material incubation period 0.5 minute-12 hours, centrifugal, the collection supernatant liquor;
2) centrifugal rear enrichment material and elutriant incubation period are 0.5 minute-12 hours, and be centrifugal, collects supernatant liquor;
3) repeating step 2) hatching, centrifugal and collection process 1-10 time.
8. according to the described phosphorylated peptide method for separating and concentrating of claim 1 or 3, it is characterized in that:
Anti-phase when adopting/after the enrichment of strong cation exchange mixed mode material goes out the monophosphate peptide, employing contains volumetric concentration 60-90% acetonitrile, methyl alcohol or the ethanolic soln of 20-200mM ammonium formiate, ammonium acetate or potassium primary phosphate, pH 2-6, with the non-phosphorylating peptide wash-out on enrichment material out.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864897A (en) * | 2014-04-03 | 2014-06-18 | 中国人民解放军防化学院 | Method for purifying phosphorylated eledoisin |
| CN104250298A (en) * | 2013-06-25 | 2014-12-31 | 浙江华谱新创科技有限公司 | Efficient separation purification method for exenatide |
| CN105866317A (en) * | 2016-03-31 | 2016-08-17 | 南方科技大学 | Proteome reactor integrating protein pretreatment and polypeptide high-pH value reverse phase classification and application thereof |
| CN105890960A (en) * | 2016-05-06 | 2016-08-24 | 同济大学 | Pre-separation method of proteins |
| WO2018126524A1 (en) * | 2017-01-04 | 2018-07-12 | 陕西慧康生物科技有限责任公司 | Method for purifying areginine essence |
| CN109956992A (en) * | 2017-12-14 | 2019-07-02 | 复旦大学 | A kind of preparation and application of the magnetic Nano material of the fixed bimetallic ion in surface |
| CN110567786A (en) * | 2019-08-06 | 2019-12-13 | 中南大学 | Spatial resolution enrichment purification sampling method for mass spectrometry imaging |
| CN111808164A (en) * | 2019-04-12 | 2020-10-23 | 中国科学院大连化学物理研究所 | Phosphorylated protein enrichment method based on multi-hydrogen bond responsive polymer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104250298A (en) * | 2013-06-25 | 2014-12-31 | 浙江华谱新创科技有限公司 | Efficient separation purification method for exenatide |
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| CN105866317A (en) * | 2016-03-31 | 2016-08-17 | 南方科技大学 | Proteome reactor integrating protein pretreatment and polypeptide high-pH value reverse phase classification and application thereof |
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| CN109956992A (en) * | 2017-12-14 | 2019-07-02 | 复旦大学 | A kind of preparation and application of the magnetic Nano material of the fixed bimetallic ion in surface |
| CN111808164A (en) * | 2019-04-12 | 2020-10-23 | 中国科学院大连化学物理研究所 | Phosphorylated protein enrichment method based on multi-hydrogen bond responsive polymer |
| CN110567786A (en) * | 2019-08-06 | 2019-12-13 | 中南大学 | Spatial resolution enrichment purification sampling method for mass spectrometry imaging |
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Application publication date: 20130515 |