CN107167542A - A kind of centrifugal device for being enriched with and separating for phosphoeptide - Google Patents
A kind of centrifugal device for being enriched with and separating for phosphoeptide Download PDFInfo
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- CN107167542A CN107167542A CN201710506881.8A CN201710506881A CN107167542A CN 107167542 A CN107167542 A CN 107167542A CN 201710506881 A CN201710506881 A CN 201710506881A CN 107167542 A CN107167542 A CN 107167542A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/80—Fraction collectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a kind of centrifugal device for being enriched with and separating for phosphoeptide.The centrifugal enriching apparatus includes enrichment pipette tips, separation pipette tips and fraction collector；It is enriched with pipette tips and is filled with SPE diaphragm A and titanium dioxide filler, covered with eight alkyl films on SPE diaphragm A, liquid outlets of the SPE diaphragm A close to enrichment pipette tips, separate and SPE diaphragm B and C18 reverse phase filler is filled with pipette tips, covered with octadecyl film on SPE diaphragm B, liquid outlets of the SPE diaphragm B close to separation pipette tips；Enrichment pipette tips may be disposed in separation pipette tips；Enrichment pipette tips and separation pipette tips can coordinate with fraction collector.The present invention passes through TiO2It is enriched with pipette tips and C18Reverse phase filler separates pipette tips and organically combines the enrichment of phosphoeptide and reverse phase separation, has effectively simplified experimental procedure, has reduced sample loss；Extensive, the phosphoric acid peptide analysis of trace clinical sample are highly suitable for, there is good application potential in the enrichment research of protein group posttranslational modification.
Description
Technical field
The present invention relates to a kind of centrifugal device for being enriched with and separating for phosphoeptide, belong to analytical chemistry field.
Background technology
Phosphorylation is one of most common posttranslational modification of protein, is referred to by protein kinase catalysis in substrate protein white matter ammonia
Covalently bound phosphate groups are added on base acid residue.Its back reaction is the dephosphorylation process by protein phosphatase enzymatic.
Protein kinase and phosphatase work independently, and in balance regulatory protein matter function.There are some researches show by gene code
Albumen in, about 30% can occur phosphorylation.This reversible process of the phosphorylation and dephosphorylation of protein almost adjusts raw
All processes of life activity, including the propagation of cell, development and break up, molecular recognition and signal transduction, nervous activity, muscle
Shrink, metabolism, tumour occurs etc..Protein phosphorylation was in the Phoebus by Rockefeller Institute of Medicine in 1906
Levene reports that he is found that the livetin of phosphorylation first.However, until 1954, the Phosphorylation events of protein were
Found by Burnett and Kennedy reports.Nineteen fifty-five Fisher and Krebs disclose reversible Phosphorylation events in biological modulated
Importance in section mechanism, and therefore obtain Nobel's physiology and Medicine.Due to protein phosphorylation have it is quite varied
And significant physiological significance, therefore turn into one of focus important in proteomics field on the research of phosphorylation.Mass spectrum
It is current protein group posttranslational modification identification research most strong instrument.But at present using biological mass spectrometry as main research hand
The phosphorylation proteomics research of section also faces a series of technological challenges：Phosphorylating protein content pole in vivo
Low, research is thought, typically in a certain specific time, the protein of phosphorylation for the overall protein matter of organism,
Content is only 2~3%；In Mass Spectrometer Method, substantial amounts of non-phosphorylating peptide fragment the signal of phosphated peptide section can be interfered and
Suppress；Phosphated peptide section has elecrtonegativity, under the positive ion mode that mass spectral analysis is commonly used, and ionization is difficult, and signal can be by
Suppress.These problems all bring no small difficulty to the scale identification of phosphated peptide section.Thus enter to more complicated sample
Before row mass spectral analysis, specific enrichment first is carried out to phosphated peptide section, its content in the sample is improved, and remove non-phosphorus
The interference of sour peptide, is just particularly important for the sensitivity for analysis and identification scale for improving phosphoeptide.The phosphorus commonly used at present
Sour peptide enrichment method mainly has metal oxide affinity chromatography (MOAC), solid metallic ion affinity chromatography (IMAC), ion to hand over
Colour changing spectrum (SAX/SCX) etc..The principle of metal oxide affinity chromatography is to utilize the reversible of transition metal oxide and phosphate radical
With reference to selective enrichment phosphated peptide section, conventional metal oxide has titanium dioxide (TiO2), zirconium dioxide (ZrO2) etc..Two
Titanium oxide is widely applied because its concentration effect is good, cost is relatively low.But it still be present：(1) sample needed for
Product initial amount is big, and (2) experimental procedure is cumbersome, extremely wastes time and energy, (3) sample analysis flux finite, (4) enrichment qualification result
Reappearance it is poor.Further, since limitation of the biological mass spectrometry instrument used at present in terms of sweep speed, after enrichment
It is multiple cuts that phosphoeptide sample, which is generally required by reversed phase chromatography separation, is further entered again after the complexity of reduction sample
Row mass spectral analysis, so as to effectively improve the identification scale of phosphoeptide.But reverse-phase chromatography pillar peptide widely used at present
Section clastotype, sample losses are larger, and need to coordinate liquid chromatograph device to use, and sample processing throughput is limited.
The content of the invention
It is an object of the invention to provide a kind of centrifugal enriching apparatus for being enriched with and separating for phosphoeptide, with it is quick,
It is easy to operate, be easy to trace sample analyze and high-throughout feature, can the multiple samples of parallel processing.
The centrifugal enriching apparatus provided by the present invention for being enriched with and separating for phosphoeptide, including enrichment pipette tips, separation
Pipette tips and fraction collector；
It is filled with the enrichment pipette tips on SPE diaphragm A and titanium dioxide filler, the SPE diaphragm A
Covered with eight alkyl films, liquid outlets of the SPE diaphragm A close to the enrichment pipette tips；
SPE diaphragm B and C18 reverse phase filler (octadecylsilane bonded silica is filled with the separation pipette tips
Glue), covered with octadecyl film on the SPE diaphragm B, the SPE diaphragm B is close to the separation pipette tips
Liquid outlet；
The enrichment pipette tips may be disposed in the separation pipette tips, i.e., coordinate in series；
The enrichment pipette tips and the separation pipette tips can coordinate with the fraction collector.
In above-mentioned centrifugal enriching apparatus, the enrichment pipette tips and the separation pipette tips are pipettor gun head；
The enrichment pipette tips can use the pipette tips of the different sizes, preferably 200 μ L specifications such as 10 μ L, 200 μ L or 1000 μ L；
The separation pipette tips can use the pipette tips of the different size such as 10 μ L or 200 μ L, preferably 10 μ L specifications.
In above-mentioned centrifugal enriching apparatus, one layer of eight alkyl film, every layer described eight are set in the enrichment pipette tips
The thickness of alkyl film is 0.2mm~0.5mm；
The consumption of the titanium dioxide filler can be 1~10mg.
In above-mentioned centrifugal enriching apparatus, 3 layers of octadecane basement membrane are set in the separation pipette tips, described in every layer
The thickness of octadecane basement membrane is 2mm~3mm；
The consumption of the C18 reverse phase fillers can be 1~10mg.
In above-mentioned centrifugal enriching apparatus, the SPE diaphragm A and the SPE diaphragm B can be used
Empore extract diaphragm, its be using polytetrafluoroethylene (PTFE) (PTFE) fiber membrane as skeleton, between fiber have using reverse phase silica gel as
Matrix or with polystyrene-divinylbenzene (PS-DVB) be matrix SPE absorbent particles.The Empore extractions diaphragm
Size dimension has difference, and thickness is about<1mm.
In above-mentioned centrifugal enriching apparatus, the enrichment pipette tips and the separation pipette tips are evaporated by adapter with described
Point collector coordinates, and the adapter can be a cylinder, specifically can be using the cylinder that internal diameter is not waited up and down.
In above-mentioned centrifugal enriching apparatus, the fraction collector can be centrifuge tube, and such as EP is managed, optional 600 μ L,
The different size such as 1500 μ L and 2000 μ L, preferably 1500 μ L.
The centrifugal enriching apparatus of the present invention can be used for enrichment and separating phosphoeptide, by the enrichment pipette tips and the separating gun
Head series connection, loading, cleaning, elution and the separation of sample are carried out with centrifugation, i.e., completes the richness of phosphoeptide in one apparatus
Collection and reverse phase separation, so that the two be organically combined, have not only simplified experimental procedure, have reduced sample loss, and reduce reality
The labor intensity tested；And the centrifugal enrichment and separation device can be used cooperatively with desk centrifuge, a parallel processing 24
Sample, substantially increases sample process flux, is highly suitable to be applied for extensive, the phosphoric acid peptide analysis of trace clinical sample.
Enrichment and separating phosphoeptide can be used for as steps described below：
(1) enrichment for adding the protein digestion product containing phosphoeptide into the centrifugal enriching apparatus
In pipette tips, then enrichment pipette tips and the fraction collector are coordinated, by the fraction collector be placed in a centrifuge into
Row centrifuge, will the phosphoeptide be enriched in the titanium dioxide filler；
(2) the enrichment pipette tips are arranged in the separation pipette tips, then received the separation pipette tips and the cut
Storage coordinates, and carries out gradient elution using the alkaline eluant of different ethane nitrile contents, gradient is collected by the fraction collector
What is eluted flows through liquid, that is, realizes the separation to the phosphoeptide.
In step (1), centrifuged using desk centrifuge, phosphoeptide is remained in the titanium dioxide filler, non-phosphoric acid
Peptide is flowed into the fraction collector with liquid is flowed through；The liquid that flows through in the fraction collector is collected, repeatedly loading 3 times, so that
Reach the purpose of abundant enrichment acid peptide；Centrifuged after adding cleaning fluid in the enrichment pipette tips, cleaning removal is adsorbed in TiO2
On non-phosphopeptide.
, will the enrichment pipette tips, the separation pipette tips and fraction collector series connection in step (2)；By enrichment
Phosphoeptide is eluted in the separation pipette tips, while utilizing C18The reverse phase filler phosphoeptide different to hydrophobicity is in ethane nitrile content
The difference of reserve capability, reverse phase separation is realized to phosphoeptide in different eluents.
As enrichment and separating mouse hepatic protein enzymolysis product in phosphoeptide when, nitrile, lactic acid, water and three can be used
Fluoroacetic acid (64:20:12:4, v/v) mixed liquor sample dissolution；Acetonitrile, water and trifluoroacetic acid (80 can be used:15:5, v/v)
Mixed liquor be used as cleaning fluid.
Phosphoeptide sample after above-mentioned enrichment, separation can be directly used for liquid chromatograph mass spectrography identification, it is not necessary to
Desalination pretreatment is carried out to sample, the sample losses that cumbersome pre-treatment operation is caused are reduced.Meanwhile, high pH reverse phase separations
The low pH reverse phase separations used during with liquid chromatograph mass spectrography have good orthogonality, can effectively reduce enriched product
Complexity, improve phosphoeptide Mass Spectrometric Identification effect.In addition, the cut cross-combining after fractionation can be retained chromatogram
The mixing of peptide fragment that behavior differs greatly, effectively improve fractionation with the identification of following liquid-phase chromatography-mass spectroscopy chromatographic isolation it is orthogonal
Property.
The present invention has advantages below：
1) TiO is passed through2It is enriched with pipette tips and C18Reverse phase filler separates pipette tips by the enrichment of phosphoeptide and the organic knot of reverse phase separation
Close, effectively simplified experimental procedure, reduced sample loss；
2) device uses high pH reverse phase separations pattern, it is to avoid in conventional SCX separation, peptide fragment desalination and lyophilized etc.
A series of cumbersome laboratory operating procedures and sample losses；
3) desk centrifuge that the device can be commonly used with laboratory is used cooperatively, and the upper of sample is carried out by centrifugation
Sample, cleaning, elution and separation, reduce the labor intensity and interference from human factor of experiment, while up to 24 samples can be realized
Parallel processing, greatly improve sample processing throughput and analysis reappearance；
4) device is simply easily made, and cost is low；
5) device be highly suitable to be applied on a large scale, the phosphoric acid peptide analysis of trace clinical sample, protein group translation
There is good application potential in the enrichment research modified afterwards.
Brief description of the drawings
Fig. 1 is the structural representation of the enrichment pipette tips of centrifugal enriching apparatus of the invention.
Fig. 2 is the structural representation of the separation pipette tips of centrifugal enriching apparatus of the invention.
Fig. 3 is the structural representation of the fraction collector of centrifugal enriching apparatus of the invention.
Schematic diagram when Fig. 4 coordinates for the enrichment pipette tips of centrifugal enriching apparatus of the invention with fraction collector.
Fig. 5 connects when coordinating for enrichment pipette tips, the separation pipette tips of centrifugal enriching apparatus of the invention with fraction collector
Schematic diagram.
Fig. 6 is the experiment flow that centrifugal enriching apparatus of the invention is applied to mouse liver proteins phosphoeptide concentration and separation
Figure.
Fig. 7 is the phosphoeptide qualification result for once testing three cuts carried out using centrifugal enriching apparatus of the invention
Wei Entu.
Fig. 8 is the Wei Entu for the phosphoeptide qualification result tested in triplicate using centrifugal enriching apparatus of the invention.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The centrifugal enriching apparatus that the present invention is provided includes enrichment pipette tips, separation pipette tips and fraction collector, wherein, it is rich
Collect the structural representation of pipette tips as shown in figure 1, it uses specification for 200 μ L pipette tips, filled with 1 layer of covering in enrichment pipette tips
There are the SPE diaphragm 1 (Empore, diameter 47nm) and 2mg titanium dioxide fillers 2 of eight alkyl films, and covered with eight alkyl
Liquid outlet of the SPE diaphragm 1 of film close to pipette tips.Separate pipette tips structural representation as shown in Fig. 2 it use specification for
SPE diaphragm 3 (Empore, diameter had three layers and covered with octadecane basement membrane is set in 10 μ L pipette tips, separation pipette tips
47nm) with 3mg C18 reverse phase fillers 4, and the SPE diaphragm 3 covered with octadecane basement membrane is close to the liquid outlet of pipette tips.Evaporate
Divide the structural representation of collector as shown in figure 3, it uses specification for 1.5mL EP pipes, provided with adapter 5, use at its mouth of pipe
Coordinate in being enriched with pipette tips and separating pipette tips.The schematic diagram being enriched with when pipette tips coordinate with fraction collector is as shown in figure 4, enrichment
Schematic diagram when pipette tips, separation pipette tips and fraction collector three series connection coordinate is as shown in Figure 5.
When being applied to mouse liver proteins phosphoeptide concentration and separation using the above-mentioned centrifugal enriching apparatus of the present invention, it can press
Carried out according to the flow shown in Fig. 4：
(1) mouse liver proteins are extracted：The liver of 10 week old C57BL/6 mouse is taken in EP pipes, adds what is configured
Urea Lysis liquid (9M urea is dissolved in 20mM HEPES, adds appropriate protease inhibitors and inhibitors of phosphatases), uses high pass
Sample is placed on ice after amount tissue grinder disrupting tissue, Protein Extraction is carried out using Ultrasonic Cell Disruptor, takes supernatant.
(2) preparation of mouse liver proteins peptide hydrolysis solution：1mg mouse orgoteins are taken, one is diluted with Urea Lysis liquid
Times, dithiothreitol (DTT) (DTT, final concentration 4.5mM) is added in the solution, and 55 DEG C of metal baths are incubated 30min.Cooled on ice is to room
Wen Hou, adds iodoacetamide (IAA, final concentration 10mM), fully mixes, room temperature lucifuge stands 30min.After diluted protein solution,
By trypsase and protein 1:100 (w/w) add trypsase, are placed in after being digested overnight in 37 DEG C of insulating boxs, use Sep-
Pak C18 desalinations pillars (Waters) are by the desalination of product peptide fragment and freeze.Enzymolysis product with 200 μ L sample solutions (acetonitrile/lactic acid/
Water/trifluoroacetic acid, 64:20:12:4, v/v) dissolve.
(3) material clean and activation：200 μ L cleaning fluid (acetonitrile/waters/trifluoroacetic acid, 80 are used respectively:15:5, v/v)
With 200 μ L sample solutions (acetonitrile/lactic acid/water/trifluoroacetic acid, 64:20:12:4, v/v) TiO is cleaned2Be enriched with pipette tips, 500g from
The heart；Respectively using the 200 pure acetonitriles of μ L and the concentrated ammonia liquors of 200 μ L 15% balance C18Separate pipette tips, 500g centrifugations.
(4) loading is enriched with：By TiO2Enrichment pipette tips are fixed on fraction collector (as shown in Figure 2), and peptide fragment solution is turned
Move to enrichment pipette tips, 500g centrifugations.The liquid that flows through in fraction collector is collected, loading is repeated, is enriched with 3 times altogether.
(5) clean：Use 200 μ L cleaning fluid (acetonitrile/waters/trifluoroacetic acid, 80:15:5, v/v) cleaning removes non-phosphoric acid
Peptide, 500g centrifugations, is repeated 3 times.
(6) elute, separate：By TiO2It is enriched with pipette tips insertion C18Separate in pipette tips, by the two series combination, and be fixed on
In new fraction collector (as shown in Figure 3).Sequentially add each 200 μ L of gradient eluent：1) 15% concentrated ammonia liquor, 500g centrifugations；
2) 2% acetonitrile+98% (15% concentrated ammonia liquor), 500g centrifugations；3) 5% acetonitrile+95% (15% concentrated ammonia liquor), 500g centrifugations；4)
8% acetonitrile+92% (15% concentrated ammonia liquor), 500g centrifugations；5) 10% acetonitrile+90% (15% concentrated ammonia liquor), 500g centrifugations；6)
40% acetonitrile+60% (15% concentrated ammonia liquor), 500g centrifugations.That collects gradient elution respectively flows through liquid, merges two-by-two：2%+8%
Acetonitrile, 5%+10% acetonitriles, 0+40% acetonitriles, -80 DEG C of preservations after freezing.
(7) Mass Spectrometer Method：By 3 cuts after enrichment with 20 μ L water/formic acid (90:10, v/v) dissolve, take 10 μ
L, carries out Mass Spectrometric Identification.Mass Spectrometry Conditions：Cation scan pattern；Electron spray voltage is 2.2kV, and first mass spectrometric resolution ratio is
120000, one-level AGC are 5.0e5, and maximum ion injection length is 50ms, and full scan mass charge ratio range is 300~1400；Two
The selection of level mass spectrometry precursor ion uses data dependence pattern, and HCD collision energies are set as that 32%, AGC is 1.0e2, maximum ion
Injection length is 250ms, and the dynamic exclusion time is set to 18s, and mass spectrum acquisition time is 78min.
(8) storehouse is searched：Proteome Discoverer1.4 softwares search storehouse analysis, and data are entered with Mascot search engines
Row retrieval, proteolytic cleavage type selecting trypsin, it is allowed to which maximum peptide fragment leakage enzyme site number is set to 2, Primary search mass deviation
≤ 15ppm, secondary search mass deviation≤0.5Da, fixation is modified to Carbamidomethyl (C), variable to be modified to
Oxidation (M), Acetyl (protein N-term), Phosphorylation (ST) and Phosphorylation (Y).
The horizontal false positive rate of peptide fragment (FDR)≤1%.
(9) phosphoeptide qualification result：Three cuts, which amount to, identifies 10793 phosphorylation sites, and correspondence 7333 is non-superfluous
Remaining phosphoeptide, enrichment selectivity is 75.8%.The phosphoeptide identification quantity and enrichment selectivity of three cuts are shown in Table 1, Wei Entu
See Fig. 7.Without fraction seperation under similarity condition, directly carry out Mass Spectrometric Identification and can only obtain 3405 phosphoeptides, enrichment selectivity
For 74.0%, illustrate that the fractionation strategy can significantly improve the identification scale of phosphoeptide on the premise of enrichment selectivity is ensured.
The peptide fragment that cut cross-combining after fractionation can be differed greatly chromatogram retention behavior is mixed, and effectively improves fractionation and follow-up
The orthogonality of chromatographic isolation in liquid chromatography-mass spectrography identification, more can make peptide fragment be evenly distributed on whole color than merging using order
Compose in the range of gradient elution, make full use of mass spectrographic sweep time.Using the device, 3 repetition experiments are identified respectively
7333,7587,7962 phosphoeptides, Wei Entu are shown in Fig. 8.Enrichment selectivity is respectively 73.1%, 73.5% and
69.1%.Wherein 50.2% phosphoeptide is at least accredited in testing twice, fully proves it in complex samples phosphorylation
Application value in analysis.
The phosphoeptide identification number and enrichment selectivity of 1 three cuts of table
Claims (9)
1. a kind of centrifugal enriching apparatus for being enriched with and separating for phosphoeptide, including enrichment pipette tips, separation pipette tips and cut are received
Storage；
Be filled with the enrichment pipette tips on SPE diaphragm A and titanium dioxide filler, the SPE diaphragm A covered with
Eight alkyl films, the SPE diaphragm A close to it is described enrichment pipette tips liquid outlet,
Be filled with the separation pipette tips on SPE diaphragm B and C18 reverse phase filler, the SPE diaphragm B covered with
Octadecane basement membrane, liquid outlets of the SPE diaphragm B close to the separation pipette tips；
The enrichment pipette tips may be disposed in the separation pipette tips；
The enrichment pipette tips and the separation pipette tips can coordinate with the fraction collector.
2. centrifugal enriching apparatus according to claim 1, it is characterised in that：The enrichment pipette tips and the separation pipette tips
It is pipettor gun head.
3. centrifugal enriching apparatus according to claim 1 or 2, it is characterised in that：One layer is set in the enrichment pipette tips
The eight alkyl film；
The consumption of the titanium dioxide filler is 1~10mg.
4. the centrifugal enriching apparatus according to any one of claim 1-3, it is characterised in that：Set in the separation pipette tips
Put 3 layers of octadecane basement membrane；
The consumption of the C18 reverse phase fillers is 1~10mg.
5. the centrifugal enriching apparatus according to any one of claim 1-4, it is characterised in that：The rule of the separation pipette tips
Lattice are less than the specification of the enrichment pipette tips.
6. the centrifugal enriching apparatus according to any one of claim 1-5, it is characterised in that：The enrichment pipette tips and institute
Separation pipette tips are stated by adapter and the fraction collector to coordinate.
7. the centrifugal enriching apparatus according to any one of claim 1-6, it is characterised in that：The fraction collector is
Centrifuge tube.
8. application of the centrifugal enriching apparatus in enrichment and separating phosphoeptide any one of claim 1-7.
9. a kind of method being enriched with separating phosphoeptide, comprises the following steps：
(1) the protein digestion product containing phosphoeptide is added to centrifugal enrichment any one of claim 1-7 and filled
In the enrichment pipette tips put, then the enrichment pipette tips and the fraction collector are coordinated, by the fraction collector
Be placed in a centrifuge and be centrifuged, will the phosphoeptide be enriched in the titanium dioxide filler；
(2) the enrichment pipette tips are arranged in the separation pipette tips, then by the separation pipette tips and the fraction collector
Coordinate, gradient elution is carried out using the alkaline eluant of different ethane nitrile contents, gradient elution is collected by the fraction collector
Flow through liquid, that is, realize the separation to the phosphoeptide.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110128499A (en) * | 2019-04-28 | 2019-08-16 | 北京谷海天目生物医学科技有限公司 | Method, detection and the quantitative method of phosphated peptide section enrichment |
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