CN107297086B - A kind of preparation of organic whole pillar and organic whole pillar and application - Google Patents
A kind of preparation of organic whole pillar and organic whole pillar and application Download PDFInfo
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Abstract
The present invention relates to a kind of preparations of Ti (IV)-IMAC organic whole pillar, and are used for the phosphorylation proteomics analysis of micro biological sample.The organic whole column material is polymethacrylate high polymer, and pore structure rich in, specific surface area is high, and the deposition activity site of titanium ion is more；At the same time, due to its hydrophilic material surface, powerful accumulation ability has been shown in terms of phosphoeptide enrichment.It is used for the phosphorylation proteomics analysis of 5g HeLa cell, has averagely identified the nonredundancy phosphorylation site more than 1000 or more, and is enriched with specificity up to 92.5%.Importantly, Ti (IV)-IMAC organic whole pillar preparation is simple, compares and fill pillar, it can only need several minutes of just preparation completions by the Raolical polymerizable of photoinduction；And the preparation without carrying out plug, largely reduce the time of enrichment material.
Description
Technical field
The invention belongs to proteomics research direction phosphorylation proteomics technical fields, and in particular to be applied to phosphorus
Acidizing protein group sample treatment.
Background technique
As important protein post-translational modification, protein phosphorylation plays most important in cell metabolism adjusting
Effect.Abnormal phosphorylation modification and many lysises is closely bound up, such as malignant tumour etc..Currently based on shotgun
Proteome analysis strategy is the main method in phosphorylation proteomics research.In this approach, protein sample
Product are digested first as polypeptide, after being enriched with by phosphoeptide, progress LC-MS/MS analysis.Since the height of phosphorylation modification is dynamic
Therefore state range, the low abundance of phosphorylated protein and the poor ionising effect of phosphated peptide section influence entire phosphorylation egg
The accuracy of white matter group credit analysis and the committed step of sensitivity are exactly the specific enrichment process of phosphoeptide.
Currently, immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOC), are two kinds main
Phosphoeptide enrichment means, wherein to belong to titanium dioxide (TiO2) and Ti (IV)-IMAC micro-sphere material be most widely used.It is close
Nian Lai, filling centrifugation pillar is widely used in micro phosphorylating protein group credit analysis, this is because it has
The advantages that operation automation, sample loss are few and no sample pollutes.But this method needs to prepare volume at the tip of centrifugation pillar
Outer plug closes pillar, unrestrained to prevent the material of filling from overflowing.Currently, absorbent cotton and C8 material are two kinds of masters
It is used for the material of plug preparation, but absorbent cotton easy absorbent solution in sample handling processes leads to higher centrifugation negative pressure,
And C8 material is also easy to produce non-specific adsorption (document 1.Zhu, the J.et al.Centrifugation of common peptide fragment
assisted microreactor enables facile integration of trypsin digestion,
hydrophilic interaction chromatography enrichment,and on-column
deglycosylation for rapid and sensitive N-glycoproteome
Analysis.Anal.Chem.84,5146-5153 (2012) document 2.Zhou, H.；Ye,M.et al.Robust
phosphoproteome enrichment using monodisperse microsphere–based immobilized
titanium(IV)ion affinity chromatography.Nat.Protoc.8,461-480(2013).)。
Therefore, the small major gene of centrifugation based on integral material its with wider pH tolerance range and excellent permeability,
It is attracted attention in terms of micro-example analysis.Krenkova et al. is prepared for organic whole based on methacrylate
Body pillar then further modifies the surface (text of Monolithic Columns using ferric oxide nano particles or hydroxyapatite nanoparticles
Offer 3.Krenkova, J.et al.Nanoparticle-modified monolithic pipette tips for
Phosphopeptide enrichment.Anal.Bioanal.Chem.405,2175-2183 (2013)), successfully from α-
It is enriched with out corresponding phosphoeptide in the pancreatin enzymolysis liquid of casein and beta-casein, still, the enrichment of complex proteins sample is real
It tests and does not carry out but, this is most likely because the Monolithic Columns lack enough enrichment specificity.Deng et al. utilizes DOPA
Amine can autohemagglutination generate poly-dopamine property, immobilized Ti (IV) ion on the catechol group of poly-dopamine, to prepare
IMAC Zip Tip entirety pillar (document 4.Yan, Y.H.et al.Facile synthesis of Ti is obtained4+-
immobilized Fe3O4@polydopamine core-shell microspheres for highly selective
Enrichment of phosphopeptides.Chem.Commun.49,5055-5057 (2013) document 5.Yan, Y.H.；
Zheng,Z.F.et al.Hydrophilic polydopamine-coated graphene for metal ion
immobilization as a novel immobilized metal ion affinity chromatography
Platform for phosphoproteome analysis.Anal.Chem.85,8483-8487 (2013) document 6.Shi,
C.Y.；Deng,C.H.et al.Immobilized metal ion affinity chromatography ZipTip
pipette tip with polydopamine modification and Ti4+immobilization for
selective enrichment and isolation of phosphopeptides.Talanta 143,464-468
(2015)), four endogenous phosphoeptides are successfully enriched with out from human serum sample.But report that poly-dopamine lacks in document
Weary enough chemical stabilities, at the same time, the Zip Tip pillar of commercialization are expensive, increase the use of the pillar at
This.Therefore, so far, the centrifugal enrichment pillar based on Monolithic Columns is not yet in the phosphoprotein of complex biological sample
It is applied in group credit analysis.
A kind of preparation of Ti (IV)-IMAC organic whole pillar is realized in invention, and is used for the phosphorus of micro-example
Acidizing protein group credit analysis, obtains efficient phosphoeptide concentration effect.Organic whole pillar preparation is simple, it is only necessary to several points
The Raolical polymerizable of clock can be completed；And it is not necessarily to the preparation of plug, when largely reducing the preparation of enrichment material
Between.
Summary of the invention
The purpose of the present invention is to provide a kind of fast and efficiently phosphoeptide beneficiation technologies Ti (IV)-IMAC organic wholes
Pillar, makes that it is suitable for the researchs of micro phosphorylation proteomics.
Ti (IV)-IMAC organic whole pillar proposed by the present invention has deposition activity site abundant and hydrophily
Material surface, the accumulation ability of efficient phosphoeptide may be implemented；And preparation is simple, time-consuming short, it can be achieved that phosphorylated protein
The quick analysis of matter group.
Specific step is as follows:
(1) mixed solution of methanol and 1,4-butanediol, volume ratio 1:2-2:1 are configured；
(2) methanol/1,4-butanediol mixed solution configuration benzophenone solution obtained using above-mentioned steps (1), matter
Amount volume fraction is 0.05-0.20g/mL；
(3) 5 μ L-20 μ L of benzophenone solution obtained in above-mentioned steps (2) is taken, 20 μ L GE Loader Tip are added to
In pipette tips (German Eppendorf company), be placed in ultraviolet generator (XL-1500A, Spectronics company, New York,
USA in), irradiation reaction 5-20 minutes under 365nm length ultraviolet light；Hexichol in centrifugation removal GE Loader tip pipette tips
Ketone solution；
(4) it repeats the above steps (3) process 1-4 time；
(5) 2 μ L-4 μ L polymer fluids are taken, is added in step (4) in ready GE Loader tip pipette tips, is placed in purple
In outer generator (XL-1500A, Spectronics company, New York, USA), reaction is irradiated under 365nm length ultraviolet light
5-20 minutes, obtain phosphate centrifugation pillar；
The volume of polymer fluid solvent forms are as follows: 12%-15%EGMP (ethylene glycol metering system phosphate),
40.0-49.0%DMSO (dimethyl sulfoxide), 7.0-10.0%DMF (dimethylformamide), 32.0-35.0% dodecanol；
Solute is 0.05-0.20g/mL Bis (methylene-bisacrylamide)；
(6) 40-60mg/mL titanium sulfate aqueous solution is configured, it is small to be added to the phosphate centrifugation prepared in above-mentioned steps (5)
In column, centrifugation reaction 10-20 minutes, discard the liquid being centrifugated out under 4000-8000g revolving speed, repeat aforesaid operations 2-4
It is secondary；
(7) wash water solution 1 is configured, wherein the volume fraction of acetonitrile is 20-40%, the volume fraction of trifluoroacetic acid is
0.1-0.3%, surplus are water, and the pillar that centrifuge washing step (6) obtains under 4000-8000g revolving speed is discarded and is centrifugated out
Liquid；It repeats aforesaid operations process 4-6 times；
(8) wash water solution 2 is configured, wherein the molar concentration of sodium chloride is 150-300mM, surplus is water, in 4000-
The pillar that centrifuge washing step (7) obtains under 8000g revolving speed, discards the liquid being centrifugated out；Repeat aforesaid operations process 4-6
It is secondary；
(9) it is washed with deionized the centrifugation pillar in above-mentioned steps (8), the centrifuge washing under 4000-8000g revolving speed,
Discard the liquid being centrifugated out；It repeats aforesaid operations process 4-6 times, it is small to obtain required product Ti (IV)-IMAC organic whole
Column.
Ti (IV)-IMAC organic whole pillar that will prepare in above-mentioned steps (9), for complicated peptide fragment example enrichment analysis
Specific step is as follows:
(1) loading buffer aqueous solution is configured, wherein the volume fraction of acetonitrile is the volume fraction of 70-90%, trifluoroacetic acid
It is water for 5-7%, surplus；
(2) using the loading buffer aqueous solution in above-mentioned steps (1), acidification balances Ti (IV)-IMAC organic whole pillar,
Centrifugation discards liquid therein；It repeats aforesaid operations process 2-4 times；
(3) it is mixed using the loading buffer aqueous solution in above-mentioned steps (1) with complicated peptide fragment sample, volume ratio 1:2-
2:1, and be transferred in Ti (IV)-IMAC organic whole pillar, the centrifugal enrichment under 4000-8000g revolving speed is centrifugated out
Liquid is again transferred in Ti (IV)-IMAC organic whole pillar, is repeated aforesaid operations process 1-3 times；
(4) wash water solution 1 is configured, wherein the volume fraction of acetonitrile is 40-60%, the volume fraction of trifluoroacetic acid is
5-7%, sodium chloride molar concentration be 150-300mM, surplus is water；Centrifuge washing step (3) under 4000-8000g revolving speed
Obtained pillar, discards cleaning solution；It repeats above-mentioned centrifuge washing operating process 1-3 times；
(5) wash water solution 2 is configured, wherein the volume fraction of acetonitrile is 20-40%, the volume fraction of trifluoroacetic acid is
0.1-0.3%, surplus are water；The pillar that centrifuge washing step (4) obtains under 4000-8000g revolving speed, discards cleaning solution；Weight
It is above-mentioned centrifuge washing operating process 1-3 times multiple；
(6) configuration elution aqueous solution, wherein the volume fraction of ammonium hydroxide is 5-20%, surplus is water；Turn in 4000-8000g
The pillar that speed lower centrifuge washing step (5) obtains, collects eluent；It repeats above-mentioned centrifugation elution action process 1-3 times, merging is washed
De- liquid obtains product.
Ti (IV)-IMAC organic whole pillar is quickly analyzed for phosphorylation proteomics, corresponding phosphorus can be obtained
The qualification result of sour peptide and phosphorylation site, the result can be used for posttranslational modification proteome analysis.
Advantages of the present invention:
Ti (the IV)-IMAC organic whole pillar has clear advantage: preparation is simple, time-consuming short, phosphoeptide enrichment effect
Rate it is high, it can be achieved that phosphorylation proteomics high throughput analysis.It compares and fills pillar, it can pass through the freedom of photoinduction
Base polymerization reaction quickly prepares completion, and the preparation without carrying out plug.Pass through the phosphorylation of beta-casein and BSA enzymolysis liquid point
Analysis, it was demonstrated that the centrifugation pillar has excellent bioaccumulation efficiency and higher specificity, and this is mainly attributed to its enrichment abundant
Active site and its hydrophilic material surface.It is used for the phosphorylation proteomics analysis of 5 μ g HeLa cells, is reflected altogether
The fixed phosphorylation site to 1185 high confidences, and it is enriched with specificity up to 92.5%.Result above confirms Ti (IV)-
IMAC organic whole pillar provides a strong tool for micro Phospoprotein group credit analysis.
Detailed description of the invention
With reference to the accompanying drawing and embodiment the present invention is described in further detail:
Fig. 1 is the preparation flow figure of Ti (IV) the organic whole pillar analyzed for phosphorylation proteomics.It is every small
2 μ L-4 μ L polymer fluids are added in column.Phosphate organic whole pillar is made by Raolical polymerizable first, further by it
It is chelated with the titanium ion of tetravalence, Ti (IV) organic whole pillar is finally made.
Fig. 2 is the scanning electron microscope diagram of Ti (IV) organic whole pillar.(A) figure clearly shows organic whole column
Microscopic appearance between material and polypropylene tube wall.(B) figure clearly shows the pattern of microcellular structure.
Fig. 3 is the MALDI TOF-MS analysis of spectra of beta-casein enzymolysis liquid.(A) be to 100fmol enzymolysis liquid directly into
Row analysis；It (B) is analyzed after digesting liquid enrichment to 100fmol；It (C) is to 10fmol enzymatic hydrolysis liquid enrichment analysis；It (D) is pair
5fmol digests liquid enrichment analysis.Asterisk (*) indicates that phosphated peptide section, pound sign (#) indicate dephosphorylation peptide fragment.
Fig. 4 is the MALDI TOF MS analysis of spectra that beta-casein enzymolysis liquid and BSA digest liquid mixture.Beta-casein enzyme
The molar ratio for solving liquid and BSA enzymolysis liquid is respectively (A) 1:500；(B)1:1000.Asterisk (*) indicates phosphated peptide section, pound sign (#)
Indicate dephosphorylation peptide fragment.
Fig. 5 is that the phosphorylation proteomics of HeLa cell sample are analyzed.(A) it is identified under different proteins applied sample amount
Nonredundancy phosphoeptide number.(B) histogram is the ratio of the mono-phosphorylated peptide and multi-phosphopeptide that identify in every group of sample, point
Line chart is its enrichment specificity.Each group of sample has all carried out mass spectrum three times and has repeated to test.Error line refers to standard deviation.
Fig. 6 is the number of the phosphorylation site identified in 5 μ g HeLa cell proteins.Each protein applied sample amount is flat
Row has carried out technology three times and has repeated to test.
Specific embodiment
Embodiment 1
The preparation process of Ti (IV)-IMAC organic whole pillar
As shown in Figure 1, molten using the methanol that volume ratio is 1:1/1,4-butanediol solution preparation 0.10g/mL benzophenone
Liquid.It takes 10 μ L benzophenone solution to be added in 20 μ L GE Loader tip (German Eppendorf company), is placed in ultraviolet hair
In raw device (XL-1500A, Spectronics company, New York, USA), 15 points of the irradiation reaction under 365nm length ultraviolet light
Clock, to which after reaction, centrifugation removes the benzophenone solution in GE Loader tip；Aforesaid operations are repeated three times, with abundant
Activate polypropylene pillar.The composition for preparing polymer fluid is as follows: 13.4%EGMP (ethylene glycol metering system phosphate),
0.10g/mL Bis (methylene-bisacrylamide), 45.0%DMSO (dimethyl sulfoxide), 8.3%DMF (dimethylformamide),
33.3% dodecanol；After taking 3 μ L polymer fluid ultrasonic degassing 10 minutes to exclude oxygen, the polypropylene for being added to above-mentioned activation is small
In column, it is placed in irradiation reaction 15 minutes under 365nm length ultraviolet line.It is to be polymerized after the reaction was completed, rinsed using methanol organic whole
To remove unreacted residue in pillar, centrifugal device is made of scapus material centrifugation pillar and two centrifuge tubes, wherein
Tip of the centrifuge tube of one 600 μ L for fixed centrifugation pillar, the centrifuge tube of another 2mL is used as centrifugation support and sample is received
Collection.The polypropylene pillar for having grafted phosphate organic whole column material as a result, is prepared.Use scanning electron microscope
(SEM, Zeiss supra55, Jena, Germany) can obtain the microscopic appearance of organic whole column material in the centrifugation pillar.Match
Make 100 μ L 50mg/mL titanium sulfate aqueous solutions, be added to it is above-mentioned prepare phosphate centrifugation pillar in, under 5000g revolving speed from
The heart reacts 15 minutes, discards the liquid being centrifugated out, repeats aforesaid operations 3 times, makes on titanium ion and organic whole column material
Phosphate group sufficiently chelate.Then using washing molten aqueous 1 (30%ACN/0.1%TFA, v/v), under 5000g revolving speed from
The heart washs the pillar at least 5 times, then with (the 200mM NaCl aqueous solution) centrifuge washing of wash water solution 2 pillar 3 times, with abundant
Remove pillar on large excess of titanium sulfate, be finally washed with deionized 3 times with remove previous step washing in remaining salt from
Son.
As shown in Fig. 2, we can be clearly seen that the table of organic whole column material by electron scanning micrograph
Face microscopic appearance.Fig. 2 .A is clearly shown key and situation between phosphate organic whole column material and polypropylene surface,
It does not occur any gap between the two, shows that phosphate organic whole column material is the table for closely grafting in polypropylene pillar
On face.Therefore, we are it can be found that the organic whole pillar can prepare tired from plug compared with filling is centrifuged pillar
It is difficult and worried, greatly simplify the preparation process of centrifugation pillar.At the same time, the scanning electron microscope of bigger amplification factor is shone
Piece (Fig. 2 .B) illustrates the cell morphology of organic whole column material.We can be found that the organic whole column material aperture is abundant,
And mostly macropore, aperture are about 5 μm.This characteristic keep away the organic whole column pillar can in the preprocessing process of sample
Exempt from very high centrifugation negative pressure occur；At the same time, macroporous structure abundant corresponds to the higher specific surface area of material, can be sudden and violent
Expose more phosphate active group, also therefore can key and more titanium ion, and this will greatly facilitate realization efficiently it is fast
The phosphoeptide enrichment of speed.More importantly the organic whole column material chemical stability is high, strong acid, strong alkali environment can tolerate,
Meet the requirement of phosphoeptide enrichment, because the enrichment and elution of phosphoeptide are usually in strong acid, highly basic in phosphoeptide enrichment process
It is carried out under the conditions of property.
Detailed process of Ti (the IV)-IMAC organic whole pillar for phosphoeptide enrichment analysis
Before phosphoeptide enrichment, first using sample-loading buffer (80%ACN, 6%TFA aqueous solution) acidification balance Ti
(IV)-IMAC organic whole pillar.First by protein enzyme solution and isometric sample-loading buffer (80%ACN, 6%TFA)
It is uniformly mixed, and is transferred in organic whole centrifugation pillar, centrifugal enrichment 30 minutes, are centrifugated out under the revolving speed of 3000g
Liquid be again transferred in Ti (IV)-IMAC organic whole pillar, repeat aforesaid operations process 2 times.Then, successively with washing
Aqueous solution 1 (50%ACN, 6%TFA, 200mM NaCl) and wash water solution 2 (30%ACN, 0.1%TFA) difference centrifuge washing
Twice, holding revolving speed is 5000g to pillar, every time washing 15 minutes, to remove the non-specific adsorption of common peptide fragment and wash molten
Salt ion in liquid 1.Finally, eluting the phosphated peptide section being adsorbed on pillar using 100 μ L, 10% ammonium hydroxide twice, merging is washed
De- liquid obtains product, and room temperature freeze-drying is stored in -30 DEG C for use.
Embodiment 2
Operating process is same as above Ti (IV)-IMAC organic whole pillar for being enriched with the detailed process of analysis, by what is prepared
Enrichment of Ti (the IV)-IMAC organic whole pillar for phosphorylated protein beta-casein is analyzed.By the pancreas of 100fmol beta-casein
Protease hydrolyzed liquid carries out the enrichment of phosphated peptide section, after completely removing the non-specific adsorption on material, with 10% ammonia
Aqueous solution elutes phosphated peptide section, and 0.5 μ L eluent point therein is taken to carry out MALDI-TOF MS analysis to MALDI target on piece.
As control experiment, the pancreatin enzymolysis liquid of the 100fmol beta-casein not Jing Guo phosphoeptide enriching step carries out in parallel
MALDI-TOF MS analysis.
As shown in Fig. 3 .A, in the mass spectrogram without the sample of enriching step, high-intensitive non-phosphopeptide Duan Feng is occupied
Entire spectrogram, can not prepare the signal peak for identifying its phosphoeptide, and a large amount of existing non-phosphorylating peptide fragments of this explanation seriously press down
The ionization of phosphated peptide section is made.But in contrast, after the enrichment of Ti (IV)-IMAC organic whole pillar, spectrum
Only occurs the mass spectra peak of clearly phosphoeptide and its dephosphorylation counterpart in figure, the signal peak of non-phosphorylating peptide fragment is almost complete
Portion disappears (Fig. 3 .B), this demonstrate that the efficiently concentrating ability of Ti (IV)-IMAC organic whole pillar.Herein on basis, we
The organic whole pillar is further investigated to the detection sensitivity of beta-casein enzymolysis liquid, as shown in Fig. 3 .C and 3.D, this has
Machine entirety pillar can successfully be enriched to the phosphoeptide in 10fmol and 5fmol beta-casein enzymolysis liquid, show that the pillar can be real
The sample analysis of existing fmol rank.
Embodiment 3
Operating process is same as above detailed process of Ti (the IV)-IMAC organic whole pillar for phosphoeptide enrichment analysis, by β-
Casein enzymolysis liquid and BSA enzymolysis liquid are mixed with different mol ratio (500:1 and 1000:1, mol/mol) to simulate complex sample,
Further assess the specificity of Ti (IV)-IMAC organic whole pillar.
As shown in figure 4, only occurring in MALDI spectrogram under the conditions of non-phosphorylating peptide fragment (BSA enzymolysis liquid) is large excess of
The clearly mass spectra peak of phosphoeptide and its dephosphorylation counterpart, even if rubbing when beta-casein enzymolysis liquid and BSA enzymolysis liquid
When you are than reaching 1000:1, the interference signal of non-phosphorylating peptide fragment is still unobvious, this demonstrate that Ti (IV)-IMAC organic whole
The high enrichment specificity of pillar.
Embodiment 4
Operating process is same as above detailed process of Ti (the IV)-IMAC organic whole pillar for phosphoeptide enrichment analysis, by it
Enrichment for actual sample HeLa cell sample is analyzed.It is enriched with analysis one respectively with Ti (IV)-IMAC organic whole pillar
The HeLa cell protein of serial different quality digests liquid, is 10 μ g, 25 μ g, 50 μ g and 100 μ g, the phosphoric acid that will be enriched to respectively
Peptide sample carries out LC-MS/MS analysis.
Fig. 5 .A gives the number of the nonredundancy phosphoeptide identified respectively in each sample.From 10 μ g HeLa cell eggs
About 1300 nonredundancy phosphated peptide sections can be identified in white matter, with the increase of sample detection quality, the phosphoric acid that identifies
The quantity of peptide is consequently increased, and identifies about 2000 nonredundancy phosphated peptide sections, 50 μ g in 25 μ g HeLa cell proteins
About 2300 are identified in HeLa cell protein, finally, in 100 μ g applied sample amount, the identification number of phosphoeptide has reached steady
Determine state, can equally identify about 2300 nonredundancy phosphated peptide sections.This demonstrate the small pillar heights of Ti (IV)-IMAC organic whole
The accumulation ability of effect can be completely applied to the phosphorylation proteomics analysis of practical biological sample.
Further count the ratio of the mono-phosphorylated peptide fragment and multi-phosphopeptide section that identify respectively in every group of sample.Such as figure
Shown in 5.B, when applied sample amount is 10 μ g and 25 μ g, the ratio of mono-phosphorylated peptide fragment is all 75% or so, and when applied sample amount increases
When to 50 μ g and 100 μ g, the ratio of mono-phosphorylated peptide fragment is reduced significantly to 50% or so.This is because when sample applied sample amount compared with
When few (10 μ g), which there is enough enrichment capacity to go to adsorb mono-phosphorylated peptide fragment and multi-phosphopeptide simultaneously
Section.However, enrichment material has progressivelyed reach the state of its saturation, simultaneously because its limited enrichment with the increase of applied sample amount
Capacity just will appear competitive Adsorption between mono-phosphorylated peptide fragment and multi-phosphopeptide section.It is readily apparent that multi-phosphopeptide section
It is stronger with the binding ability of the titanium ion on adsorbent material due to including multiple phosphate radicals, so as to by enrichment material
Material preference it is enriched with.Therefore, we can be found that Ti (IV)-IMAC organic whole pillar in 25 μ g HeLa cell eggs
Reach saturation when white applied sample amount, when applied sample amount increases to 50 μ g and 100 μ g, which has been in oversaturated
State.Therefore the enrichment capacity of Ti (the IV)-IMAC organic whole pillar is 25 μ g.
The enrichment specificity of Ti (the IV)-IMAC organic whole pillar also demonstrates this point simultaneously.Work as protein example
When amount is 10 μ g, enrichment specificity up to 95%；When sample applied sample amount increases to 25 μ g and 50 μ g, under absorption specificity is small size
Falling, but is still up to 90% or so, this mainly has benefited from the higher bioaccumulation efficiency of the material, but when protein example amount swashs
When increasing to 100 μ g, enrichment specificity is then reduced significantly to only 75% or so.It is organic that Ti (the IV)-IMAC is demonstrated again
The enrichment capacity of whole pillar is 25 μ g.
Embodiment 5
Operating process is same as above detailed process of Ti (the IV)-IMAC organic whole pillar for phosphoeptide enrichment analysis, Ti
(IV)-IMAC organic whole pillar is used for the analysis of micro-example.Enrichment analysis is carried out to 5 μ g HeLa cell proteins enzymatic hydrolysis liquid,
Obtained phosphoeptide sample is subjected to analysis detection by autosampler loading to mass spectrograph, while carrying out technology three times and repeating
Test.
Fig. 6 gives the phosphorylation site number of identified nonredundancy in experiment every time.Technology repeats in experiment three times,
1087,1043 and 1060 nonredundancy phosphorylation sites are identified from 5 μ g HeLa cell proteins respectively, wherein having respectively
802,767 and 772 sites be high confidence phosphorylation site (Class I, i.e. localization probability >
0.75, score difference > 5).This is in current document report, from 5 μ g complex cell samples, in conjunction with automatic sampling
Standard LC MS/MS analysis method, the best result identified.It is 92.7% (RSD=to the enrichment specificity of phosphoeptide
0.2%).So high enrichment specificity also shows the efficiently concentrating ability of the organic whole pillar simultaneously.
The present invention is a kind of preparation of Ti (IV)-IMAC organic whole pillar, and is used for the phosphorus of micro biological sample
Acidizing protein group credit analysis.The organic whole column material is polymethacrylate high polymer, pore structure rich in,
Specific surface area is high, and the deposition activity site of titanium ion is more；At the same time, due to its hydrophilic material surface, in phosphoeptide richness
Collection aspect has shown powerful accumulation ability.It is used for the phosphorylation proteomics analysis of 5 μ g HeLa cells, it is average
The nonredundancy phosphorylation site more than 1000 or more has been identified, and has been enriched with specificity up to 92.5%.Importantly, should
The preparation of Ti (IV)-IMAC organic whole pillar is simple, compares and fills pillar, it can be anti-by the free radical polymerization of photoinduction
Several minutes of just preparation completions should only be needed；And the preparation without carrying out plug, when largely reducing the preparation of enrichment material
Between.
Claims (5)
1. a kind of preparation method of organic whole pillar, it is characterised in that:
Specific step is as follows:
(1) mixed solution of methanol and 1,4-butanediol, volume ratio 1:2-2:1 are configured；
(2) methanol/1,4-butanediol mixed solution configuration benzophenone solution obtained using above-mentioned steps (1), mass body
Fraction is 0.05-0.20 g/mL；
(3) 5 μ L-20 μ L of benzophenone solution obtained in above-mentioned steps (2) is taken, 20 μ L GE Loader Tip are added to
It in pipette tips, is placed in ultraviolet generator, irradiation reaction 5-20 minutes under 365 nm length ultraviolet light；Centrifugation removal GE
Benzophenone solution in Loader tip pipette tips；
(4) it repeats the above steps (3) process 1-4 time；
(5) 2 μ L-4 μ L polymer fluids are taken, is added in step (4) in ready GE Loader tip pipette tips, is placed in ultraviolet
In generator (XL-1500 A, Spectronics company, New York, USA), the irradiation reaction 5- under 365 nm wavelength
20 minutes, obtain phosphate centrifugation pillar；
The volume of polymer fluid solvent forms are as follows: 12%-15% EGMP(ethylene glycol metering system phosphate), 40.0-
49.0% DMSO(dimethyl sulfoxide), 7.0-10.0% DMF(dimethylformamide), 32.0-35.0% dodecanol；Solute is
0.05-0.20 g/mL Bis(methylene-bisacrylamide)；
(6) 40-60 mg/mL titanium sulfate aqueous solution is configured, the phosphate centrifugation pillar prepared in above-mentioned steps (5) is added to
Interior, centrifugation reaction 10-20 minutes, discard the liquid being centrifugated out under 4000-8000 g revolving speed；Repeat aforesaid operations process 2-
4 times；
(7) wash water solution 1 is configured, wherein the volume fraction of acetonitrile is 20-40%, the volume fraction of trifluoroacetic acid is 0.1-
0.3%, surplus is water, and the pillar that centrifuge washing step (6) obtains under 4000-8000 g revolving speed discards the liquid being centrifugated out
Body；It repeats aforesaid operations process 4-6 times；
(8) wash water solution 2 is configured, wherein the molar concentration of sodium chloride is 150-300 mM, surplus is water, in 4000-8000
The pillar that centrifuge washing step (7) obtains under g revolving speed, discards the liquid being centrifugated out；It repeats aforesaid operations process 4-6 times；
(9) the centrifugation pillar in above-mentioned steps (8) is washed with deionized, the centrifuge washing under 4000-8000 g revolving speed discards
The liquid being centrifugated out；It repeats aforesaid operations process 4-6 times, obtains required product Ti (IV)-IMAC organic whole pillar.
2. organic whole pillar prepared by a kind of claim 1.
3. a kind of application of organic whole pillar as claimed in claim 2, it is characterised in that: organic whole pillar is used for complexity
The analysis of peptide fragment example enrichment, the specific steps are as follows:
(1) loading buffer aqueous solution is configured, wherein the volume fraction of acetonitrile is 70-90%, the volume fraction of trifluoroacetic acid is 5-
7%, surplus is water；
(2) using the loading buffer aqueous solution in above-mentioned steps (1), acidification balances Ti (IV)-IMAC organic whole pillar, from
The heart discards liquid therein；It repeats aforesaid operations process 2-4 times；
(3) it is mixed using the loading buffer aqueous solution in above-mentioned steps (1) with complicated peptide fragment sample, volume ratio 1:2-2:1,
And it is transferred in Ti (IV)-IMAC organic whole pillar, the centrifugal enrichment under 4000-8000 g revolving speed, the liquid being centrifugated out
Body is again transferred in Ti (IV)-IMAC organic whole pillar；It repeats aforesaid operations process 1-3 times；
(4) configure wash water solution 1, wherein the volume fraction of acetonitrile is 40-60%, the volume fraction of trifluoroacetic acid be 5-7%,
The molar concentration of sodium chloride is 150-300 mM, surplus is water；Centrifuge washing step (3) obtains under 4000-8000 g revolving speed
Pillar, discard cleaning solution；It repeats above-mentioned centrifuge washing operating process 1-3 times；
(5) wash water solution 2 is configured, wherein the volume fraction of acetonitrile is 20-40%, the volume fraction of trifluoroacetic acid is 0.1-
0.3%, surplus is water；The pillar that centrifuge washing step (4) obtains under 4000-8000 g revolving speed, discards cleaning solution；In repetition
State centrifuge washing operating process 1-3 times；
(6) configuration elution aqueous solution, wherein the volume fraction of ammonium hydroxide is 5-20%, surplus is water；Under 4000-8000 g revolving speed
The pillar that centrifuge washing step (5) obtains collects eluent；It repeats above-mentioned centrifugation elution action process 1-3 times, merges eluent
Obtain product.
4. the application of organic whole pillar according to claim 3, it is characterised in that:
The complexity peptide fragment sample is the protein of one of cell sample, blood sample or tissue sample or two kinds or more
One of trypsin digestion liquid of mixture or two kinds or more.
5. the application of organic whole pillar according to claim 3, it is characterised in that: being applied to the organic whole pillar can
The analysis for realizing phosphorylation proteomics, can obtain the qualification result of corresponding phosphoeptide and phosphorylation site, which can
To be used for posttranslational modification proteome analysis.
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| CN113318715B (en) * | 2020-02-28 | 2022-04-08 | 中国科学院大连化学物理研究所 | Phosphorylated peptide adsorbent and preparation and application thereof |
| CN113138249B (en) * | 2021-04-12 | 2021-11-23 | 北京蛋白质组研究中心 | Micro-sample metabolome, proteome and phosphoproteome multi-group chemical analysis method based on micropore array chip |
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| CN1811406A (en) * | 2006-01-26 | 2006-08-02 | 复旦大学 | Method for fast enriching trace polypeptide and protein and realizing identification |
| CN101396650A (en) * | 2007-09-26 | 2009-04-01 | 中国科学院大连化学物理研究所 | Titanium ion fixation affinity chromatography material and preparation and use thereof |
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| CN1811406A (en) * | 2006-01-26 | 2006-08-02 | 复旦大学 | Method for fast enriching trace polypeptide and protein and realizing identification |
| CN101396650A (en) * | 2007-09-26 | 2009-04-01 | 中国科学院大连化学物理研究所 | Titanium ion fixation affinity chromatography material and preparation and use thereof |
| CN104195042A (en) * | 2014-07-27 | 2014-12-10 | 北京化工大学 | Preparation method and application of nano material monolithic column immobilized enzyme biological micro-reactor |
| CN104594021A (en) * | 2015-02-13 | 2015-05-06 | 武汉大学 | HSAB theory based sulfhydryl cotton post-modification material as well as preparation and application thereof |
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