CN110501409A - A kind of protein desalination method and its application - Google Patents
A kind of protein desalination method and its application Download PDFInfo
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- CN110501409A CN110501409A CN201910804753.0A CN201910804753A CN110501409A CN 110501409 A CN110501409 A CN 110501409A CN 201910804753 A CN201910804753 A CN 201910804753A CN 110501409 A CN110501409 A CN 110501409A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
Abstract
The present invention relates to be related to a kind of protein desalination method and its application.The protein desalination method is the following steps are included: (1) first uses TFA soluble protein quality sample, centrifuging and taking supernatant；(2) Zip Tip is soaked with ACN, then is balanced with TFA；(3) using wetting in step (2) and the Zip Tip after balance, pressure-vaccum makes the sample in step (1) at least through adsorption column 2 times for several times；It is rinsed again with flushing liquor TFA；(4) elution peptide fragment is used again；(5) SpeedVac drains sample.Desalination method provided by the present invention, it can be adapted for being enriched with by phosphorylation modification in the protein of processing, overcome the problem that desalination method protein losses are big in the prior art, extend the application range of phosphorylation modification protein group, leap of the phosphorylation modification protein group from non-marked to label is realized, phosphorylation modification beneficiation technologies and labelling technique are preferably combined togather.
Description
Technical field
The present invention relates to a kind of protein desalination methods, and in particular to a kind of protein desalination method and its application.
Background technique
Isotope labelling is opposite and absolute quantitation (Isobaric Tags for Relative and Absolute
Quantitation, abbreviation iTRAQ) and TMT (Tandem Mass Tag) be quantitative with relatively broad two kinds of labels at present
Protein technique, by conjunction with peptide fragment N-terminal group (can also be combined with lysine side chain amino groups, in addition to this two
A binding site, also there are many binding sites for TMT different type reagent), realize the label to polypeptide, and pass through different molecular weight
Reagent method that separate sources sample is marked realize the target that different sample room protein compare, in conjunction with report ion
The same peptide fragment of calculated by peak area same protein different sample rooms ratio, to realize the quantitative comparison of albumen.
Phosphorylation modification is that under natural conditions, one kind that protein occurs is reversibly modified, is frequently accompanied by protein kinase
Effect, since phosphorylation modification level is very low, so being enriched with out the destination protein of generation phosphorylation modification from holoprotein very
Important, the property based on phosphate anion, enrichment method is based on metal ion and metal oxide, such as TiO2、Ti-
IMAC, Fe-IMAC etc..Phosphated peptide section enrichment method can convenient and efficient the peptide fragment by phosphorylation modification from whole peptide fragments point
It separates out and, be used for proteome analysis.In phosphorylation modification peptide fragment beneficiation technologies, sample passes through cracking, reductive alkylation, enzyme
It after solution processing, is directly enriched with, in this process, sample is not by removing salt treatment, due to being enriched with obtained sample
Seldom, desalination method is not appropriate for this kind of sample to amount in conventional group, this is because conventional desalination method has biggish loss, and
Peptide fragment after enrichment is very micro, however, labelled reagent cannot not used directly by the sample except salt treatment
Label, is only used for non-marked proteomics research.
Summary of the invention
The present invention for the technical problems in the prior art, provides a kind of protein desalination method and it is answered
With.The desalination method can be used for the desalination of trace protein, can meet the research of labelled protein group.
The present invention is achieved through the following technical solutions above-mentioned technical purpose:
The present invention provides a kind of protein desalination methods, comprising the following steps:
(1) TFA soluble protein quality sample, centrifuging and taking supernatant are first used；
(2) Zip Tip is soaked with ACN, then is balanced with TFA；
(3) using wetting in step (2) and the Zip Tip after balance, pressure-vaccum makes the sample in step (1) at least for several times
Pass through adsorption column 2 times；It is rinsed again with flushing liquor TFA；
(4) elution peptide fragment is used again；
(5) SpeedVac drains sample.
Wherein, in step (1) TFA and protein example usage ratio are as follows: every 100 μ g protein 40-60 μ l0.2%
The dissolution of TFA solution.
Wherein, in step (1) TFA final concentration of 0.1%-1%, pH < 4.
Wherein, the mass concentration of the ACN in step (2) is 40-60%, and the mass concentration of TFA is 0.05-0.15%.
Wherein, the concentration of flushing liquor TFA is 0.05-0.15% in step (3).
Wherein, eluent is the mixed liquor containing 40-60%ACN and 0.05-0.15%TFA in step (4).
Preferably, a kind of protein desalination method provided by the present invention, comprising the following steps:
(1) 0.2%TFA solution sample dissolution, the final concentration of 0.1-1% of TFA, pH < 4, centrifuging and taking supernatant are used；
(2) Zip Tip is soaked with 50%ACN fountain solution repeatedly, then is balanced with 0.1%TFA；
(3) using wetting in step (2) and the Zip Tip after balance, pressure-vaccum makes the sample in step (1) at least for several times
Pass through adsorption column 2 times；It is rinsed again with 0.1%TFA flushing liquor；
(4) peptide fragment is eluted with the mixed liquor containing 50%ACN and 0.1%TFA again；
(5) SpeedVac drains sample.
It is highly preferred that a kind of protein desalination method provided by the present invention, comprising the following steps:
(1) 0.2%TFA solution sample dissolution, the final concentration of 0.1-1% of TFA, pH < 4, centrifuging and taking supernatant are used；
(2) 15 wetting Zip Tip of 50%ACN fountain solution pressure-vaccum are used repeatedly, then are washed with 0.1%TFA and blown 10 balances；
(3) using wetting in step (2) and the Zip Tip after balance, pressure-vaccum makes the sample in step (1) at least for several times
Pass through adsorption column 2 times；It is blown and beaten 5 times and is rinsed with 0.1%TFA flushing liquor again；
(4) 15 elution peptide fragments are blown and beaten repeatedly with the mixed liquor containing 50%ACN and 0.1%TFA again；
(5) SpeedVac drains sample.
The present invention also provides a kind of application of above-mentioned protein desalination method, protein is enriched with by phosphorylation modification peptide
After processing, using removing salt treatment, in the investigative technique for labelled protein group.
Wherein, the investigative technique of labelled protein group is that iTRAQ or TMT marks quantitative protein group technology.
Wherein, TFA is trifluoroacetic acid, and ACN is acetonitrile.
The present invention will first be dissolved to the sample peptide fragment of desalination 0.1%TFA, centrifugation removes undissolved impurity, then by Zip
Tip is activated except salt plug with 50% acetonitrile solution, then is achieved the purpose that preferably adsorb peptide fragment with TFA solution equilibria, then
The peptide fragment to desalination is suspended to Zip Tip again to remove on salt plug pillar, salinity and impurity are eluted by 0.1% TFA solution, finally
Peptide fragment is eluted with the TFA mixed solution of 50% ACN, 0.1%, peptide fragment is collected and drains, machine testing on mass spectrum.
The present invention has following advantageous effects:
(1) desalination method provided by the present invention can be adapted for being enriched with by phosphorylation modification in the protein of processing,
The problem that desalination method protein losses are big in the prior art is overcome, extend phosphorylation modification protein group applies model
It encloses, realizes leap of the phosphorylation modification protein group from non-marked to label, by phosphorylation modification beneficiation technologies and label skill
Art is preferably combined togather；
(2) optimization Jing Guo process, phosphorylation beneficiation technologies can expand to labelled protein from non-marked proteomics
Matter group is all greatly improved from sample size and the scope of application, extends the application scenarios of technique, is phosphoric acid
Change modification proteomics preferably serves scientific research or clinical project provides technology guarantee；
(3) since, there are experimental error and systematic error, micro desalination can preferably reduce system mistake in operating process
Difference is maintained at the variation of desalination front and back sample size in allowable range of error, the amount of each sample can be controlled in same water
It is flat；
(4) reach desalination purpose using micro desalination for several times, by removing salt treatment, then carry out the egg of iTRAQ or TMT label
The research of white matter group, allows multiple samples to be analyzed and studied in same level, wide in scientific research and clinical sample
General application.
Detailed description of the invention
Fig. 1 is the result of protein labeling efficiency in comparative example and embodiment 1；
Fig. 2 is the result of phosphorylation modification Identification of Fusion Protein number in comparative example and embodiment 1.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
Embodiment 1
It takes Arabidopsis leaf to obtain albumen through extractions such as over cleaning, homogenate, cracking, ultrasounds, carries out reduction alkyl in next step
Change, evaluated by albumen of the gel electrophoresis of protein to extraction, then digested overnight, it is rich that next day carries out phosphorylation modification peptide
Collection and micro desalination are eventually used for the label such as iTRAQ, carry out LC-MS/MS detection after classification.
It is as follows that phosphorylation modification is enriched with flow chart:
Wherein the step of micro desalination is as follows:
(1) protein example obtained after first dissolving appropriate phosphorylation enrichment with the TFA solution of 50 μ l 0.2%pH < 4,
12000rpm centrifugation 2min takes supernatant；
(2) range of pipettor is adjusted at 10 μ l, is installed Zip Tip pillar, is put into 50%ACN solution, blows repeatedly
15 wetting Zip Tip are made a call to, then the Zip Tip pillar after wetting is put into 0.1% TFA solution, are blown and beaten 10 times repeatedly
Balance Zip Tip pillar；
(3) completely logical using wetting in step (2) and 7 samples made in step (1) of Zip Tip pressure-vaccum after balance
Adsorption column is crossed, i.e., the Zip Tip pillar balanced is put into the Supernatant samples after centrifugation, blows and beats 7 times repeatedly, allows peptide fragment can be very
It is adsorbed on well on Zip Tip pillar；The peptide fragment for removing some non-phosphorylating modifications is rinsed with 0.1%TFA flushing liquor again,
Piping and druming 5 times for using 0.1%TFA flushing liquor dispense flushing liquor by every 400 μ l of pipe, each sample is independent to avoid cross contamination
With a pipe, each sample does 4 repetitions.
(4) it is eluted again with 50%ACN and 0.1%TFA mixed solution, that is, 50 μ l eluents is taken to manage to clean EP
In, it blows and beats 15 times and is eluted repeatedly, it is each to repeat elution 4 times, i.e., it is respectively eluted in 4 pipe eluents 15 times.
(5) SpeedVac drains sample.
Embodiment 2
It takes Arabidopsis leaf to obtain albumen through extractions such as over cleaning, homogenate, cracking, ultrasounds, carries out reduction alkyl in next step
Change, evaluated by albumen of the gel electrophoresis of protein to extraction, then digested overnight, it is rich that next day carries out phosphorylation modification peptide
Collection and micro desalination are eventually used for the label such as iTRAQ, carry out LC-MS/MS detection after classification.
It is as follows that phosphorylation modification is enriched with flow chart:
Wherein the step of micro desalination is as follows:
(1) first with the protein sample obtained after first dissolving the enrichment of appropriate phosphorylation with the TFA solution of 40 μ l 0.7%pH < 4
Product, 10000rpm centrifugation 5min take supernatant；
(2) range of pipettor is adjusted at 10 μ l, is installed Zip Tip pillar, is put into 40%ACN solution, blows repeatedly
12 wetting Zip Tip are made a call to, then the Zip Tip pillar after wetting is put into 0.05% TFA solution, are blown and beaten 8 times repeatedly
Balance Zip Tip pillar；
(3) completely logical using wetting in step (2) and 5 samples made in step (1) of Zip Tip pressure-vaccum after balance
Adsorption column is crossed, i.e., the Zip Tip pillar balanced is put into the Supernatant samples after centrifugation, blows and beats 5 times repeatedly, allows peptide fragment can be very
It is adsorbed on well on Zip Tip pillar；The peptide for removing some non-phosphorylating modifications is rinsed with 0.05%TFA flushing liquor again
Section, that is, piping and druming 8 times for using 0.05%TFA flushing liquor dispense flushing liquor, each sample by every 400 μ l of pipe to avoid cross contamination
It is individually managed with one, each sample does 4 repetitions；
(4) it is eluted again with 40%ACN and 0.05%TFA mixed solution, that is, 50 μ l eluents is taken to manage to clean EP
In, it blows and beats 15 times and is eluted repeatedly, it is each to repeat elution 2 times, i.e., it is respectively eluted in 4 pipe eluents 20 times；
(5) SpeedVac drains sample.
Embodiment 3
It takes human liver cancer tissue to obtain albumen through extractions such as over cleaning, homogenate, cracking, ultrasounds, carries out reduction alkyl in next step
Change, evaluated by albumen of the gel electrophoresis of protein to extraction, then digested overnight, it is rich that next day carries out phosphorylation modification peptide
Collection and micro desalination are eventually used for the label such as iTRAQ, carry out LC-MS/MS detection after classification.
It is as follows that phosphorylation modification is enriched with flow chart:
Wherein the step of micro desalination is as follows:
(1) first with the protein sample obtained after first dissolving the enrichment of appropriate phosphorylation with the TFA solution of 60 μ l 1%pH < 4
Product, 10000rpm centrifugation 8min take supernatant；
(2) range of pipettor is adjusted at 10 μ l, is installed Zip Tip pillar, is put into 60%ACN solution, blows repeatedly
18 wetting Zip Tip are made a call to, then the Zip Tip pillar after wetting is put into 0.15% TFA solution, are blown and beaten 12 times repeatedly
Balance Zip Tip pillar；
(6) completely logical using wetting in step (2) and 8 samples made in step (1) of Zip Tip pressure-vaccum after balance
Adsorption column is crossed, i.e., the Zip Tip pillar balanced is put into the Supernatant samples after centrifugation, blows and beats 8 times repeatedly, allows peptide fragment can be very
It is adsorbed on well on Zip Tip pillar；The peptide for removing some non-phosphorylating modifications is rinsed with 0.15%TFA flushing liquor again
Section, that is, piping and druming 4 times for using 0.15%TFA flushing liquor dispense flushing liquor, each sample by every 400 μ l of pipe to avoid cross contamination
It is individually managed with one, each sample does 4 repetitions；
(7) it is eluted again with 60%ACN and 0.15%TFA mixed solution, that is, 50 μ l eluents is taken to manage to clean EP
In, it blows and beats 15 times and is eluted repeatedly, it is each to repeat elution 7 times, i.e., it is respectively eluted in 4 pipe eluents 10 times；
(8) SpeedVac drains sample.
Comparative example
It takes Arabidopsis leaf to obtain albumen through extractions such as over cleaning, homogenate, cracking, ultrasounds, carries out reduction alkyl in next step
Change, evaluated by albumen of the gel electrophoresis of protein to extraction, then digested overnight, it is rich that next day carries out phosphorylation modification peptide
Collection is eventually used for the label such as iTRAQ, carries out LC-MS/MS detection after classification, comparative example is the difference from embodiment 1 is that phosphoric acid
Sample after changing enrichment is without micro except detection is directly marked in salt treatment.
The result of protein labeling efficiency is as shown in Figure 1, comparative example and phosphorylation in embodiment 1 in comparative example and embodiment 1
The result for modifying Identification of Fusion Protein number is as shown in Figure 2.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of protein desalination method, which comprises the following steps:
(1) TFA soluble protein quality sample, centrifuging and taking supernatant are first used；
(2) Zip Tip is soaked with ACN, then is balanced with TFA；
(3) using wetting in step (2) and the Zip Tip after balance for several times pressure-vaccum make the sample in step (1) at least through
Adsorption column 2 times；It is rinsed again with flushing liquor TFA；
(4) elution peptide fragment is used again；
(5) SpeedVac drains sample.
2. a kind of protein desalination method according to claim 1, which is characterized in that TFA and protein sample in step (1)
The usage ratio of product are as follows: every 100 μ g protein 40-60 μ l 0.2%TFA solution dissolves.
3. a kind of protein desalination method according to claim 1, which is characterized in that TFA's is final concentration of in step (1)
0.1%-1%, pH < 4.
4. a kind of protein desalination method according to claim 1, which is characterized in that the quality of the ACN in step (2) is dense
Degree is 40-60%, and the mass concentration of TFA is 0.05-0.15%.
5. according to right to go 1 described in a kind of protein desalination method, which is characterized in that flushing liquor TFA's is dense in step (3)
Degree is 0.05-0.15%.
6. a kind of protein desalination method according to claim 1, which is characterized in that in step (4) eluent be containing
The mixed liquor of 40-60%ACN and 0.05-0.15%TFA.
7. a kind of protein desalination method according to claim 1, which comprises the following steps:
(1) 0.2%TFA solution sample dissolution, the final concentration of 0.1-1% of TFA, pH < 4, centrifuging and taking supernatant are used；
(2) Zip Tip is soaked with 50%ACN fountain solution repeatedly, then is balanced with 0.1%TFA；
(3) using wetting in step (2) and the Zip Tip after balance for several times pressure-vaccum make the sample in step (1) at least through
Adsorption column 2 times；It is rinsed again with 0.1%TFA flushing liquor；
(4) peptide fragment is eluted with the mixed liquor containing 50%ACN and 0.1%TFA again；
(5) SpeedVac drains sample.
8. a kind of protein desalination method according to claim 1, which comprises the following steps:
(1) 0.2%TFA solution sample dissolution, the final concentration of 0.1-1% of TFA, pH < 4, centrifuging and taking supernatant are used；
(2) 15 wetting Zip Tip of 50%ACN fountain solution pressure-vaccum are used repeatedly, then are washed with 0.1%TFA and blown 10 balances；
(3) using wetting in step (2) and the Zip Tip after balance for several times pressure-vaccum make the sample in step (1) at least through
Adsorption column 2 times；It is blown and beaten 5 times and is rinsed with 0.1%TFA flushing liquor again；
(4) 15 elution peptide fragments are blown and beaten repeatedly with the mixed liquor containing 50%ACN and 0.1%TFA again；
(5) SpeedVac drains sample.
9. the application of protein desalination method as described in claim 1, which is characterized in that protein passes through phosphorylation modification peptide
After enrichment processing, using removing salt treatment, in the investigative technique for labelled protein group.
10. the application of protein desalination method according to claim 9, which is characterized in that labelled protein group is ground
Studying carefully technology is that iTRAQ or TMT marks quantitative protein group technology.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111521473A (en) * | 2020-04-28 | 2020-08-11 | 中国人民解放军军事科学院军事医学研究院 | Device for preparing proteomics micro samples |
| CN112505328A (en) * | 2020-11-02 | 2021-03-16 | 武汉金开瑞生物工程有限公司 | Isotope labeling kit and labeling method |
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| WO2001011087A2 (en) * | 1999-08-10 | 2001-02-15 | Genvec, Inc. | Method of evaluating a gene transfer vector and gene transfer vector product interactions |
| US20030143609A1 (en) * | 2000-07-06 | 2003-07-31 | Genvec, Inc | Method of identifying a gene product |
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