CN102539215A - Method for storing protein sample for proteomics at normal temperature - Google Patents
Method for storing protein sample for proteomics at normal temperature Download PDFInfo
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Abstract
The invention discloses a method for storing a protein sample for proteomics at normal temperature. The method comprises the following steps of: dissolving the protein sample into Tris- saturated phenol, putting the phenol phase into a supersaturated ammonium sulfate methanol solution to precipitate protein, and storing the precipitated protein in the supersaturated ammonium sulfate methanol solution for a long term at normal temperature, wherein the protein sample comprises a crude extract of the protein sample or a commercial protein sample. By the storing method, the protein sample can be stored for half a month at normal temperature and stored for one year and even longer at a low temperature such as 20 DEG C below zero; and 1-DE and 2-DE results show that the protein sample stored by the method has good quality and good mass spectrum compatibility, and is convenient to transport.
Description
Technical field
The invention belongs to the proteomics field, be specifically related to a kind of normal temperature and preserve the method for proteomics with protein sample.
Background technology
Laemmli used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate T4 bacteriophage coat protein (SDS-PAGE, Laemmli 1970) according to the size of molecular weight in 1970 first.On the basis of Laemmli, O ' Farrell has invented polyacrylamide two dimensional gel electrophore-sis (2-DE) technology (O ' Farrell, 1975), and this technology is applied in the proteomics research widely.In the two dimensional gel electrophore-sis technology, specimen preparation is a crucial step.The method for distilling that is applied to vegetable protein at present is a lot, and the most frequently used have TCA/ acetone precipitation (Giavalisco et al., 2003; Canovas et al., 2004), phenol is taken out method (Phe) (Hurkman and Tanak; 1986), these methods are applied in the proteomics research widely subsequently, on the basis of these methods; Through continuous improvement and optimization, people such as wang have found a kind of BPP method (Wang et al., 2007) that can well from plant tissue, extract albumen; Present this method has been applied to (Wang et al., 2009 in the proteomics research widely; Wang et al., 2011).
In proteomics research, the quality of albumen directly has influence on the result of dielectrophoresis, and albumen is prone to degraded, generally the albumen powder freeze-drying and be dissolved in urea and the lysate of thiocarbamide in use and preserve (Gorg et al., 2004; Peltier et al., 2004; Valeria et al., 2005; Bao et al., 2006; Sun et al., 2007), still, how long such albumen can not be preserved at low temperatures, preserves under the normal temperature more again, and is not easy to transportation, and this is bringing very big difficulty for the researcher aspect albumen storage and transport.
Summary of the invention
The object of the present invention is to provide a kind of normal temperature to preserve the method for proteomics with protein sample; This store method can make protein sample preserve at normal temperatures to reach two weeks, at low temperatures as can preserve below-20 ℃ 1 year, in addition more of a specified duration; Result through 1-DE and 2-DE finds; The quality of the protein sample of preserving by the inventive method is fine, and it is compatible to have good mass spectrum simultaneously, and convenient transportation.
Above-mentioned purpose of the present invention realizes through following technical scheme: a kind of normal temperature is preserved the method for proteomics with protein sample; Protein sample is dissolved in the saturated phenol of Tris-; Get phenol and place supersaturation ammonium sulfate methanol solution to be settled out albumen mutually, and the albumen that is precipitated out is carried out long preservation at normal temperatures get final product in supersaturation ammonium sulfate methanol solution.
The powder of the crude extract of protein sample optimization protein sample of the present invention or commercially available protein sample, the crude extract of this protein sample can adopt conventional methods such as BPP method from plant or animal sample, to extract.
The pH value of the saturated phenol of Tris-according to the invention is more than or equal to 7.8.
Phenol according to the invention is preferably 1: 1 with the volume ratio of supersaturation ammonium sulfate methanol solution～and 10.
The present invention is dissolved in protein sample in the saturated phenol of Tris-, gets phenol and places supersaturation ammonium sulfate methanol solution to be settled out albumen mutually, and the albumen that is precipitated out can be preserved 1～15 day in supersaturation ammonium sulfate methanol solution at normal temperatures.
The present invention is dissolved in protein sample in the saturated phenol of Tris-, gets phenol and places supersaturation ammonium sulfate methanol solution to be settled out albumen mutually, and the albumen that is precipitated out was being preserved below-20 ℃ 1～3 year in supersaturation ammonium sulfate methanol solution.
Protein sample of the present invention is pressed in the solution and can be preserved two weeks by normal temperature at saturated sulfuric acid, can preserve 1 year at-20 ℃, even more of a specified duration.In order to detect the quality of albumen,, find that the albumen quality of according to said method preserving is fine to 1-DE and 2-DE collection of illustrative plates; Can well be used in the proteomics research; It is compatible to have good mass spectrum simultaneously, no matter be to use aircraft dispatch, or train transport protein sample; Can not influence the quality of albumen, this has brought very big convenience for the transportation of albumen and preservation.
Compared with prior art, the present invention has following advantage:
(1) uses the inventive method protein sample to preserve and reach two weeks at normal temperature;
(2) use the inventive method protein sample to preserve more than 1 year at-20 ℃;
(3) sample that uses the inventive method to preserve is convenient to transportation at normal temperatures;
(4) the protein sample quality of using the inventive method to preserve is fine, the 1-DE and the 2-DE result that can obtain simultaneously;
(5) it is compatible that the sample that uses the inventive method to preserve possesses good mass spectrum simultaneously;
(6) use the inventive method in the research of proteomics, bringing into play important effect, have a extensive future.
Description of drawings
Fig. 1 is 30 ℃ of 1-DE protein graphical spectrums of placing different time sections among the embodiment 1, and wherein: B0, B1, B3, B5, B10 and B15 are respectively that bovine serum albumin (BSA) sample was placed respectively 0 day at 30 ℃, 1 day, and 3 days, 5 days, the 1-DE of 10 days and 15 days; C0, C1, C3, C5, C10 and C15 are respectively that rubber tree C-lactalbumin sample was placed respectively 0 day at 30 ℃, 1 day, and 3 days, 5 days, the 1-DE of 10 days and 15 days, M is albumen Marker;
Fig. 2 is 30 ℃ of dielectrophoresis (2-DE) protein graphical spectrums of placing different time sections C-whey among the embodiment 2, wherein: C0, C1; C3, C5, C10 and C15 are respectively that rubber tree C-lactalbumin sample was placed respectively 0 day at 30 ℃; 1 day, 3 days, 5 days; The dielectrophoresis collection of illustrative plates of 10 days and 15 days schemes wherein that arrow target point is to have done the point that mass spectrum is identified among the C15;
Fig. 3 is dielectrophoresis (2-DE) protein graphical spectrum of protein sample behind different store methods of sugarcane blade and bark among the embodiment 3, and wherein A is that the protein sample of sugarcane blade is kept at dielectrophoresis (2-DE) protein graphical spectrum in-20 ℃ the supersaturation ammonium sulfate; B is that the protein sample of sugarcane blade is preserved two months dielectrophoresis (2-DE) protein graphical spectrum in 4 ℃ of refrigerators, wherein regional a and b, and compare in e and f zone, and regional b and f are the apparent in view zones of protein sample degraded.Figure C is that the protein sample of rubber tree bark has been preserved dielectrophoresis (2-DE) protein graphical spectrum in 1 year in-20 ℃ supersaturation ammonium sulfate;
Fig. 4 is the peptide finger-print of the protein site C297 that identifies out through MALDI TOF/TOF MS among the embodiment 2; Wherein protein site C297 is excised; Analyze through MALDI-TOF/TOF with trypsin digestion; Peptide finger-print (PMF) spectrum peak is represented the intensity of different peptide sections, and the part of sequence underscoring and the portion of non-line represent peptide hop count coupling and non-coupling respectively.
Embodiment
As do not have special indicating, below the reagent or the method that are adopted be conventional reagent or conventional method, wherein w/v refers to the bulking value percentage composition, v/v refers to volumn concentration.
Bovine serum albumin(BSA) (BSA), available from BIOSHARP company, with bovine serum albumin(BSA) (BSA) powder dissolution in the saturated phenol of Tris-(pH 8.0); The saturated phenol of Tris-(pH 8.0) is available from Beijing Suo Laibao Science and Technology Ltd., and (ammonium sulfate is added in the methanol solution, makes solution reach supersaturation then phenol to be added to the supersaturation ammonium sulfate methanol solution of 5 times of volumes; Being placed on 4 ℃ spends the night subsequent use) in protein precipitation, in supersaturation ammonium sulfate methanol solution, in 30 ℃ of incubators, placed 0 day 1 day to the protein sample that has precipitated respectively; 3 days; 5 days, 10 days and 15 days, final sample all be placed on-20 ℃ subsequent use.Need to prove that this supersaturation ammonium sulfate methanol solution is all to be oversaturated under any temperature: after configuring at normal temperatures, be placed in the refrigerator about 4 ℃ and preserve, take out before the use with getting final product.
Extract the albumen in the latex of panama rubber tree C-whey with BPP method (Wang et al.2007, Wang et al.2010).Wherein, it is following from latex of panama rubber tree C-whey, to extract protein process: adopt the BPP method, add 3mL BPP protein extract (100mM EDTA, 100mM Tris (pH 8.0) in the C-whey of every 1mL; The 50mM borax, 50mM vitamin C, 1%PVPP (w/v), 1%Triton X-100 (v/v); Room temperature after 5 minutes, adds the saturated phenol of isopyknic Tris-(pH 8.0) with the whirlpool mixing then, continues the whirlpool and mixes vibration 10min, 4 ℃; Change upper strata phenol over to another centrifuge tube mutually after the 12000rpm, centrifugal 15min, to wherein adding the equal-volume protein extract, vibration 5min is mixed in the whirlpool; 4 ℃, the centrifugal 15min of 12000rpm changes upper strata phenol in another centrifuge tube mutually, to the supersaturation ammonium sulfate methanol solution protein precipitation that wherein adds 5 times of volumes; And in supersaturation ammonium sulfate methanol solution, in 30 ℃ of incubators, placed the albumen behind the supersaturation ammonium sulfate precipitation respectively 0 day, 1 day, 3 days; 5 days, 10 days and 15 days, be placed on then-20 ℃ subsequent use.
Then at 4 ℃, 15min, centrifugal under the 15000g condition, remove supernatant; Deposition is used the above methanol wash of-20 ℃ of precooling 6h once, again at 4 ℃, 15min is centrifugal under the 15000g condition; Remove supernatant, deposition is used the above washing with acetone twice of-20 ℃ of precooling 6h again, washs back 4 ℃ at every turn; The centrifugal 15min of 15000g, albumen precipitation is air-dry in room temperature, use lysate [7M urea at last; The 2M thiocarbamide, quality percentage composition 2%CHAPS (monoethanolamine propyl group dimethylamino propane sulfonic acid salt), 13mM DTT (dithiothreitol (DTT))] melt albumen more than 2 hours in room temperature.
What SDS-PAGE used is 16 centimetres of slab gels; Separation gel is that 12.5% polyacrylamide, concentrated glue are 4% polyacrylamide (Laemmli 1970) for the quality percentage composition for the quality percentage composition; Then protein sample is carried out the SDS-PAG gel electrophoresis; Electrophoresis is 4 hours under 16 ℃, 8w condition, and gel is through coomassie brilliant blue staining, and testing result is seen shown in Figure 1.
Place 0 day (Fig. 1: B0) from bovine serum albumin (BSA) sample respectively at 30 ℃; 1 day (Fig. 1: B1), 3 days (Fig. 1: B3), 5 days (Fig. 1: B5); 10 days (Fig. 1: B10) with 15 days (Fig. 1: B15) can find out on the 1-DE collection of illustrative plates; The band of bovine serum albumin is very clear, does not have diffusing phenomenon to occur, and the protein sample quality that this explanation uses the inventive method to preserve is fine.In order further to detect the inventive method, the applicant has been 1-DE with latex of panama rubber tree C-whey again, and wherein the C-lactalbumin has also been placed 0 day (Fig. 1: C0) respectively at 30 ℃; 1 day (Fig. 1: C1), 3 days (Fig. 1: C3), 5 days (Fig. 1: C5); 10 days (Fig. 1: C10) with 15 days (Fig. 1: C15), from these figure can find out that the band of C-lactalbumin mainly concentrates between the 17kDa-130kDa, what each band all divided opens very much; Do not have the band of disperse to occur, this further illustrates, the albumen that from tissue, extracts; Use method of the present invention the normal temperature preservation to reach two weeks, be convenient to transportation, to the not influence of quality of protein sample.
Embodiment 2
The pH value that is dissolved in that extracts by the method among the embodiment 1 is the latex of panama rubber tree C-lactalbumin CE in 8.0 the saturated phenol of Tris-, with the supersaturation ammonium sulfate methanol solution post precipitation of 6 times of volumes, the albumen that is precipitated out is placed respectively 0 day in 30 ℃ of incubators in supersaturation ammonium sulfate methanol solution; 1 day, 3 days, 5 days; After 10 days and 15 days, obtain the albumen in the 800 microgram C-wheys by the washing of the method among the embodiment 1 crack protein, with the linear IPG adhesive tape (GE Healthcar) of the long pH value of 24cm 4-7; Aquation is 18 hours in the aquation dish; Then use to focus on appearance (Hoefer IEF100) and accomplish the isoelectric focusing process, after the completion adhesive tape for the first time at equilibrium liquid (50mMTris-HCl, pH8.8; 6M urea; Volumn concentration 30% glycerine, quality percentage composition 2% sodium dodecylsulphonate (SDS), quality percentage composition are 0.02% bromjophenol blue) in to add the quality percentage composition be that 1% dithiothreitol (DTT) (DTT) soaked adhesive tape 15 minutes; In equilibrium liquid, add for the second time the quality percentage composition and be 4% iodoacetamide and soak adhesive tape after 15 minutes, balance finishes the back and uses ultrapure water that level pad residual on the adhesive tape is rinsed well.Adhesive tape after the balance is transferred to second upper end to vertical gel (the service property (quality) percentage composition is 12.5% polyacrylamide gel); With the agarose of quality percentage composition 1% with adhesive tape sealing; Use electrophoresis apparatus (Hoefer) to carry out the SDS-PAGE gel electrophoresis; Initial permanent power 6W/ gel electrophoresis 1h regulates permanent power 8W/ gel electrophoresis 6h afterwards to bromophenol blue indicator arrival gel bottom.Electrophoresis carries out follow-up gel-colored process after accomplishing, and the result sees Fig. 2.The albumen that from the C-whey, extracts in supersaturation ammonium sulfate methanol solution, preserve respectively 0 day (Fig. 2, C0), 1 day (Fig. 2, C1); 3 days (Fig. 2, C3), 5 days (Fig. 2, C5); 10 days (Fig. 2, C10) with 15 days (Fig. 2, two dimensional gel electrophore-sis (2-DE) collection of illustrative plates after C15) can be found out; The protein sample that uses the inventive method to preserve, it is fine that protein site gathers, and do not have conditions of streaking to occur; The proof protein sample is preserved under the method, and the quality of sample does not change, and can be used in this inventive method in the proteomics research.
In order further to confirm the MS compatibility of this inventive method; The application inventor has preserved 2-DE (Fig. 2 of 15 days latex C-whey from supersaturation ammonium sulfate methanol solution; C15) some protein sites after the dyeing have been cut on; 11 representational protein sites are identified through MALDI TOF MS, and are further confirmed that through TOF/TOF MS/MS the result sees table 1.Peptide finger-print from the mass spectrum result of the albumen of whole latex C-whey (some C297) is revealed in Fig. 4, and Search Results shows that this is esterase protein (Esterase) (table 1).As what see among Fig. 4, the peptide section that these are complementary has very low noise background, and being of high quality of peptide finger-print PMF and fragment ion spectrogram (PFF) is described.
In 11 protein sites identifying; Every kind of albumen has all obtained higher search mark; 1 protein site (some C258) is identified out to be 14-3-3 albumen, and 1 protein site (some C2004) is identified out to be latex anaphylactogen (latex protein allergen Hev b 7) (table 1).These albumen all are the distinctive albumen of C-whey in the latex; Albumen in many other latex is also by clear and definite evaluation come out (table 1); This explains the 2-DE collection of illustrative plates that albumen store method of the present invention can not only obtain; It is compatible to have good mass spectrum simultaneously, for proteomics research provides certain help.
The MALDI TOF/TOF MS/MS qualification result of C-lactalbumin in table 1 latex
aThe protein site that representative marks in Fig. 2.
bAccession number in the NCBInr database.
cThrough the protein name of MALDI TOF MS evaluation and the peptide sequence of identifying through TOF/TOF MS/MS
dIdentify theory (T.) molecular weight (kDa) and the isoelectric point (pI) of albumen
ePeptide hop count (MP) that matees among the PMF or PFF and total peptide hop count (TP) .SC identify albumen and cover the amino acid sequence scope;---refer to what not evaluation was come out
fThe search mark of PMF and PFF NCBInr.
Embodiment 3
The BPP method (Wang et al.2007) of pressing among the embodiment 1 is extracted the albumen in sugarcane blade and the rubber tree bark; Behind the supersaturation ammonium sulfate methanol solution protein precipitation with 4 times of volumes; A part of sample of sugarcane blade is placed in-20 ℃ of supersaturation ammonium sulfate methanol solutions and preserved two months; Another part sample of sugarcane blade carries out (not being stored in the supersaturation ammonium sulfate methanol solution) after the washing and cracking of albumen by the method among the embodiment 1, in 4 ℃ of refrigerators, preserved two months.The sample of preserving in-20 ℃ of supersaturation ammonium sulfate methanol solutions is also by the washing of the method among the embodiment 1 cracking.The sample of two kinds of different store methods carries out the dielectrophoresis experiment, and the result sees Fig. 3.Can find out that from the dielectrophoresis collection of illustrative plates sample is preserved 4 ℃ of refrigerators after, and dielectrophoresis (2-DE) collection of illustrative plates (Fig. 3, B) result is bad; Protein site does not separately have conditions of streaking to occur, particularly at several zone (Fig. 3; B:b and f) PD clearly, and the sample of in-20 ℃ of supersaturation ammonium sulfate methanol solutions, preserving (Fig. 3, dielectrophoresis collection of illustrative plates A) is fine; Particularly in two zones (Fig. 3, A:a and e), it is good that big protein site also gathers; There is not conditions of streaking to occur; This proof protein sample adds behind lysate and can not preserve in that 4 ℃ of refrigerators are medium-term and long-term, but but can long preservation in-20 ℃ supersaturation ammonium sulfate methanol solution, and the quality of albumen does not change.
In order further to confirm the holding time of protein sample in-20 ℃ supersaturation ammonium sulfate methanol solution; The inventor is the albumen that in rubber tree bark, extracts; In-20 ℃ supersaturation ammonium sulfate methanol solution, preserved 1 year; Press the method washing crack protein among the embodiment 1,1200 microgram albumen by the same method in front obtain the dielectrophoresis collection of illustrative plates (Fig. 3, C).From the collection of illustrative plates of rubber tree bark, can find out; It is fine that each protein site all gathers; Show no sign of conditions of streaking, protein sample of this explanation the inventive method not only can normal temperature in supersaturation ammonium sulfate methanol solution be preserved two weeks, the more important thing is that it can also can preserve 1 year even more of a specified duration at-20 ℃; And can well be used in the proteomics research the not influence of result of dielectrophoresis.
In a word, the albumen store method among the present invention has brought convenience for the albumen storage and transport, and more later proteomics research provides help.
Get commercially available animal protein sample such as the ovalbumin of chicken; Protein sample is dissolved in the pH value greater than in 7.8 the saturated phenol of Tris-; The saturated phenol of Tris-source is the same, gets phenol and places supersaturation ammonium sulfate methanol solution to be settled out albumen mutually, and the volume ratio of phenol and supersaturation ammonium sulfate methanol solution is 1: 1 and the albumen that is precipitated out preserved 10 days as 10～40 ℃ in supersaturation ammonium sulfate methanol solution at normal temperatures; Result through 1-DE and 2-DE finds; The quality of the protein sample that the method that provides by present embodiment is preserved is fine, and it is compatible to have good mass spectrum simultaneously, and convenient transportation.
Get commercially available fish protein sample; Protein sample is dissolved in the pH value greater than in 7.8 the saturated phenol of Tris-, and the source is the same in the saturated phenol of Tris-, gets phenol and places supersaturation ammonium sulfate methanol solution to be settled out albumen mutually; The volume ratio of phenol and supersaturation ammonium sulfate methanol solution is 1: 10 and the albumen that is precipitated out was preserved 15 days in supersaturation ammonium sulfate methanol solution at normal temperatures; And preserving below-20 ℃ 3 years, through result's discovery of 1-DE and 2-DE, preserved at normal temperatures 15 days; And fine in the quality of protein sample of preserving 3 years below-20 ℃, it is compatible to have good mass spectrum simultaneously.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (5)
1. a normal temperature is preserved the method for proteomics with protein sample; It is characterized in that: protein sample is dissolved in the saturated phenol of Tris-; Get phenol and place supersaturation ammonium sulfate methanol solution to be settled out albumen mutually, and the albumen that is precipitated out is carried out long preservation at normal temperatures get final product in supersaturation ammonium sulfate methanol solution.
2. normal temperature according to claim 1 is preserved the method for proteomics with protein sample, and it is characterized in that: described protein sample is the crude extract of protein sample or commercially available protein sample.
3. normal temperature according to claim 1 and 2 is preserved the method for proteomics with protein sample, it is characterized in that: pH value >=7.8 of the saturated phenol of said Tris-.
4. normal temperature according to claim 1 is preserved the method for proteomics with protein sample, and it is characterized in that: said phenol is 1: 1～10 with the volume ratio of supersaturation ammonium sulfate methanol solution.
5. normal temperature according to claim 1 is preserved the method for proteomics with protein sample; It is characterized in that: protein sample is dissolved in the saturated phenol of Tris-; Get phenol and place supersaturation ammonium sulfate methanol solution to be settled out albumen mutually, and the albumen that is precipitated out was preserved 1～15 day in supersaturation ammonium sulfate methanol solution at normal temperatures.
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| CN112321671A (en) * | 2020-11-10 | 2021-02-05 | 海南师范大学 | Method for extracting vegetable protein of rubber trees and hippocampus by phenol extraction method |
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| US20090076019A1 (en) * | 2006-10-13 | 2009-03-19 | Mount Sinai Hospital | Methods for treating neurological disorders or damage |
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