CN100580414C

CN100580414C - Method for protein gel internal enzymolysis

Info

Publication number: CN100580414C
Application number: CN200610165282A
Authority: CN
Inventors: 李银心; 王旭初
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2006-12-15
Filing date: 2006-12-15
Publication date: 2010-01-13
Anticipated expiration: 2026-12-15
Also published as: CN1987401A

Abstract

The method includes following procedures: (1) using ultra pure water to wash up gel containing target protein; dehydrating and drying gel; (2) using treatment fluid of acidized trypsin to soak dryed gel for 0.5-1.5h to absorb unwanted enzyme treatment fluid; the treatment fluid of acidized trypsin is obtained through following steps: using 80-120mM hydrochloric acid to dissolve trypsin, acidizing it for 8-12 minutes, using NH4HCO3 to adjust pH to 8.0-9.0; (3) adding buffer solution containing Ca2+, carrying out enzymolysis at 37 deg.C for 14-18h, removing gel to obtain proteolysis fluid. Reducing difficulty of operation, simplifying experimental procedure, lowering cost, the method raises efficiency of enzymolysis effectively, obtains more information of peptide segment, and higher point value of searching database, and raises percentage of coverage of sequence markedly so as to obtain better qualification result of mass spectrum finally.

Description

A kind of method of protein gel internal enzymolysis

Technical field

The present invention relates to a kind of method that is applicable to the interior enzymolysis of protein adhesive of mass spectrophotometry.

Background technology

Along with finishing of extensive biological gene group examining order, the mankind have entered the protein group epoch.At present in proteome research, mass spectrum (MS) technology is most crucial protein identification techniques (C á novas F M, ElianeD G, Recorbet G, Jorrin J, Mock H P and Rossignol M.Plant proteome analysis.Proteomics, 2004,4:285-298; Chen S and Harmon A C.Advances in plantproteomics.Proteomics, 2006,6:5504-5516).Difference according to ioning method, mass spectrum mainly is divided into substance assistant laser desorpted ionized flight mass spectrum (Matrix-assisted laserdesorption/ionization time-of-flight, MALDI TOF) and electron spray ionisation flight mass spectrum (Electrospray ionization time-of-flight, ESI-TOF) two major types (C á novas F M, Eliane D G, Recorbet G, Jorrin J, Mock H P and Rossignol M.Plant proteomeanalysis.Proteomics, 2004,4:285-298).Normally by bidirectional electrophoresis technique (two-dimensional gel electrophoresis, 2-DE) protein mixture is separated, cut the target protein point, carry out enzymolysis in the glue with trypsase (trypsin), carry out mass spectrophotometry then, obtain peptide quality fingerprinting spectrum (peptide mass fingerprinting, PMF), finally both can directly search the storehouse and identify target protein according to the PMF data, also can further obtain to search the storehouse behind the part peptide section sequence by second order ms, obtain more reliable qualification result (C á novas F M, Eliane D G, Recorbet G, Jorrin J, Mock H P andRossignol M.Plant proteome analysis.Proteomics, 2004,4:285-298; RossignolM, Peltier J B, Mock H P, Matros A, Maldonado A M, Jorr í n J V.Plant proteomeanalysis:A 2004-2006 update.Proteomics, 2006,6:5529-5548).

Which kind of mass spectrum authenticate technology no matter, enzymolysis all is a vital step in the protein adhesive.The target protein enzymolysis whether fully, what, the height of sequence coverage rate (sequence coverage) of whether special, the disconnected quantity (peptide number) of enzyme section of restriction enzyme site, and enzyme the autotomy height of degree, the power of signal to noise ratio (S/N ratio) etc., all can directly influence the result that searches the storehouse and the success ratio of Identification of Fusion Protein.Though appearance along with various advanced persons' mass spectrometer, the success ratio of Identification of Fusion Protein improves greatly, but the progress of instrument does not also have to arrive the degree that does not need to carry out enzymolysis in the glue after all, finally to obtain with a high credibility, the Identification of Fusion Protein result of good reproducibility, still depend on to a great extent and select enzyme solution (Kumarathasan P in the suitable glue for use, Mohottalage S, Goegan P and VincentR.An optimized protein in-gel digest method for reliable proteomecharacterization by MALDI-TOF-MS analysis.Analytical Biochemistry, 2005,346:85-89).

Many enzyme solutions can be used in the mass spectrum research, also obtained certain effect, but all there is certain not enough (Kumarathasan P, Mohottalage S, Goegan P and Vincent R.An optimized proteinin-gel digest method for reliable proteome characterization byMALDI-TOF-MS analysis.Analytical Biochemistry, 2005,346:85-89).The cost that adds present mass spectrum authenticate technology is still very high, and each experiment expense plenty of time of all deflorating removes relatively to grope different enzyme solutions with fund, and for the laboratory of carrying out the mass spectrum evaluation on a small scale, cost is still too high.Therefore, it is just very necessary to grope a kind of enzyme solution simple and easy to do, satisfactory for result.

Enzyme solution is that people such as Rosenfeld reports (Rosenfeld J on Analytic Biochemistry magazine in the glue early, Capdevielle J, Guillemont J C and Ferrara P.In-gel digestionof proteins for internal sequence analysis after one-or two-dimensional gelelectrophoresis.Analytical Biochemistry, 1992,203:173-179); Subsequently, method (Hellman U after several optimizations improve has been arranged again, Wernstedt C, Gonez J and Heldin C H.Improvement of an " In-Gel " Digestion Procedure for the Micropreparation ofInternal Protein Fragments for Amino Acid Sequencing.AnalyticalBiochemistry, 1995,224:451-455; Rachel R, Tracy I S, Charles M, JosephA L and Philip C A.Mass Spectrometry of Proteins Directly from PolyacrylamideGels.Anal Chem, 1996,68 (11): 1910-1917; Alan D, David C and Liang L.ProteinConcentration and Enzyme Digestion on Microbeads for MALDI-TOF Peptide MassMapping of Proteins from Dilute Solutions.Anal Chem, 2000,72 (14): 3355-3362; Volker E, Konrad B, Christine L, Hans L and Eckhard N.Improvements in Protein Identification by MALDI-TOF-MS Peptide Mapping.AnalChem, 2000,72 (13): 2741-2750; Vukic S and Jasminka G Z.Improvement ofan in-gel tryptic digestion method for matrix-assisted laserdesorption/ionization time of flight mass spectrometry peptide mapping byuse of volatile solubilizing agents.Proteomics, 2001,1:1364-1367).Though these methods by successful Application in mass spectrum research, but all exist such as various deficiencies (Kumarathasan P such as enzymolysis are incomplete, peptide section annealing efficiency is low, Mohottalage S, Goegan P and Vincent R.Anoptimized protein in-gel digest method for reliable proteomecharacterization by MALDI-TOF-MS analysis.Analytical Biochemistry, 2005,346:85-89).(2005) recently, people such as Kumarathasan are through systematic Study, reported enzyme solution (Kumarathasan P in a kind of improved glue, Mohottalage S, Goegan P and Vincent R.An optimized protein in-gel digest method for reliable proteomecharacterization by MALDI-TOF-MS analysis.Analytical Biochemistry, 2005,346:85-89), effectively improved the success ratio of Identification of Fusion Protein, the sensitivity that mass spectrum is identified has improved 6-10 doubly (for sake of convenience, below abbreviating this enzyme solution as the K method) than conventional method.

Though the K method has improved Identification of Fusion Protein confidence level and success ratio greatly than conventional enzyme solution, there are several obvious deficiencies in this method.In order to improve the specially property of restriction enzyme site, this method is adjusted to 7.0 with the pH value of final enzymolysis, thereby has effectively avoided the mistake of enzyme to cut.But bovine trypsin (Trypsin) is a kind of enzyme of alkalescence, has the highest enzyme and cut specificity (seeing Roche company product description for details) when pH7.5-9.0.Therefore, improving enzyme to neutrality and cut specificity by regulating the pH value, is to be cost with the activity that reduces the Trypsin enzyme and specificity.Therefore, people such as Kumarathasan remedies by strengthening the enzyme amount.The enzyme amount that each protein site is used reaches 500ng (Kumarathasan P, Mohottalage S, Goegan P and Vincent R.An optimizedprotein in-gel digest method for reliable proteome characterization byMALDI-TOF-MS analysis.Analytical Biochemistry, 2005,346:85-89), and usually, there is 50ng just enough.Increasing enzyme amount can improve enzyme really and cut effect, but add excessive trypsase, when causing the waste of enzyme, raising the cost (Trypsin of Roche company costs an arm and a leg), very easily cause enzyme autotomy and mistake is cut, how to eliminate autotomy peak and other assorted peak of enzyme when identifying and caused very big difficulty for the back mass spectrum.In addition, this method has also strengthened the intensity of final peptide section extracting, by progressively improving the concentration of acetonitrile in the extract (ACN), can obtain better extraction rate was acquired (Kumarathasan P, Mohottalage S, Goegan P andVincent R.An optimized protein in-gel digest method for reliable proteomecharacterization by MALDI-TOF-MS analysis.Analytical Biochemistry, 2005,346:85-89).But, when obtaining peptide section as much as possible, also the impurity in the glue is extracted together, thereby protein sample effective ion process when seriously disturbing mass spectrum to identify causes and loses the peak.Therefore adopt this method to carry out the enzymolysis experiment, have deficiencies such as enzymolysis is insufficient, the peptide section is lost seriously, the enzyme consumption big, complex operation equally, the result of final data library searching neither be too desirable.

Summary of the invention

The method that the purpose of this invention is to provide a kind of protein gel internal enzymolysis.

The method of protein gel internal enzymolysis provided by the present invention comprises the steps:

1) gel that will contain target protein washs with ultrapure water; With gel dehydration, drying;

2) with behind the trypsase working fluid immersion 0.5-1.5h of dried gel with acidifying, inhale and remove unnecessary enzyme working fluid; The trypsase working fluid of described acidifying is with 80-120mM dissolving with hydrochloric acid trypsase, acidifying 8-12 minute, uses NH again ₄HCO ₃The pH value is adjusted to the solution that 8.0-9.0 obtains;

3) adding contains Ca ²⁺Damping fluid, 37 ℃ of enzymolysis 14-18hr remove gel and obtain protein enzymatic hydrolyzate.

The concentration of hydrochloric acid step 2) is 100mM; Described NH ₄HCO ₃Concentration be 50mM; Enzyme concentration behind the described 100mM dissolving with hydrochloric acid trypsase is 0.08-0.12 μ g/ μ l, is preferably 0.1 μ g/ μ l; The trypsinase concentration of described trypsase working fluid is 8-12ng/ μ l, is preferably 10ng/ μ l.

In the described step 1), the consumption of ultrapure water is that 50-150 μ l/50 μ g contains protein gel, is preferably 100 μ l/50 μ g and contains protein gel, and wash time is 15-45min, is preferably 30min; Washing times is more than 2 times or 2 times, is preferably 2 times.

After also comprising washing in the described step 1), slough the step of Coomassie brilliant blue color in the gel with destainer; Described destainer is that 45-55% acetonitrile and quality percentage composition are 0.356-0.435%NH for containing the quality percentage composition ₄HCO ₃, the pH value is the aqueous solution of 8.0-9.0.Described destainer is preferably that to contain the quality percentage composition be that 50% acetonitrile and quality percentage composition are 0.395%NH ₄HCO ₃, the pH value is the aqueous solution of 8.0-9.0.

Soak dehydration with acetonitrile in the described step 1).

Contain Ca in the described step 3) ²⁺Damping fluid for containing 0.8-1.2mM CaCl ₂And 20-30mMNH ₄HCO ₃, the pH value is the solution of 8.0-9.0.

The described Ca that contains ²⁺Damping fluid be preferably and contain 1mM CaCl ₂With 25mM NH ₄HCO ₃, the pH value is 8.5 solution.

Method of the present invention is a kind of interior enzyme solution of protein adhesive that is suitable for the simplification of mass spectrophotometry.Method of the present invention at first, has strengthened gel detergent intensity (per 50 μ g protein gels are used the ultrapure washing twice of 80-120 μ l instead, each half an hour), can effectively remove impurity and part salt ion; Secondly, increase the step (using 100mM hcl acidifying 10 minutes) of enzyme acidification, improved tryptic enzymolysis activity effectively; Simultaneously, when pre-enzymolysis, exempted adding CaCL ₂Step (Ca ²⁺Activity for Trypsin plays an important role), and inhale and remove unnecessary enzyme liquid, the effect of autotomying of Trypsin before also formally not carrying out enzymolysis can effectively be avoided.The enzymolysis liquid that method of the present invention obtains can directly carry out mass spectrophotometry, has not only saved trivial step such as extracting of peptide section and vacuum drying, and can solve the difficult problem that the peptide section is difficult to dissolve after the vacuum drying preferably.

With salicornia europaeal Rubisco and BSA is that the experiment of research material shows, utilize K method and short cut technique enzymolysis BSA of the present invention and Rubisco albumen, with MALDI TOF enzyme analysis hydrolysis products, final by Mascot Distiller software search ncbi database, can both purchase and obtain good qualification result, score and sequence coverage rate be very high (table 1) all, illustrates that these two kinds of enzyme solutions can be used in the mass spectrum research.

The MALDI TOF data that enzyme solution and k method obtain in the glue that the present invention simplifies are finally searched the storehouse result and are comprehensively compared, and it is more more credible than the result that K method obtains that the present invention simplifies the interior enzyme solution of glue.At first, the database search score height that obtains of short cut technique of the present invention.The Rubisco albumen and the BSA score that obtain with the K method are respectively 104 minutes (Fig. 1 D) and 117 minutes (Fig. 2 D), and short cut technique score of the present invention is respectively 117 minutes (Fig. 1 E) and 148 minutes (Fig. 2 E) (table 1).Secondly, the PMF collection of illustrative plates quantity of information that short cut technique of the present invention obtains will be far away more than the K method.With short cut technique enzymolysis of the present invention, can obtain 53 (Fig. 1 C) and 70 total peptide sections (Fig. 2 C) respectively, and use the K method, obtain respectively 49 (Figure 1B) and 33 peptide sections (Fig. 2 B).In addition, short cut technique of the present invention can obviously improve the sequence coverage rate of identifying albumen.In carrying out the experiment of Rubisco albumen and BSA mass spectrum, in the short cut technique enzymolysis product of the present invention, there are 16 and 23 peptide sections must go up respectively with the sequences match in the database, coverage rate is up to 41% and 37%.Utilize the K method to carry out enzymolysis, finally can not get so high sequence coverage rate (table 1).

The mass spectrum database of information Search Results that enzyme solution obtains in the different glue of table 1. relatively

Method of the present invention is when reducing operation easier, simplification experimental procedure, reducing cost, can effectively improve enzymolysis efficiency, obtain more polypeptide segment information, obtain higher database search score value, significantly improve the sequence coverage rate, finally obtained better mass spectrum qualification result.Therefore, method of the present invention has the mass spectrum compatibility, can reduce the loss of peptide section behind the protein digestion effectively, obtains more reliable identification of proteins result, can be used as enzyme solution in a kind of effective, stable glue, is applied in the proteomics research.

Description of drawings

Figure 1A is salicornia europaeal Rubisco albumen bidimensional electrophoretic separation result

Figure 1B is with the mass spectrogram that obtains behind the K method enzymolysis salicornia europaeal Rubisco albumen

The mass spectrogram that Fig. 1 C obtains after with enzymatic isolation method enzymolysis salicornia europaeal Rubisco albumen in the glue of the present invention

Fig. 1 D searches the storehouse result for the NCBI with the mass spectrometric data that obtains behind the K method enzymolysis salicornia europaeal Rubisco albumen

Fig. 1 E searches the storehouse result for the NCBI with the mass spectrometric data that obtains behind the enzymatic isolation method enzymolysis salicornia europaeal Rubisco albumen in the glue of the present invention

Fig. 2 A is the unidirectional electrophoresis separating resulting of BSA

Fig. 2 B is with the mass spectrogram that obtains behind the K method enzymolysis BSA

Fig. 2 C is for to simplify the mass spectrogram that obtains behind the enzymatic isolation method enzymolysis BSA in the glue with the present invention

Fig. 2 D searches the storehouse result for the NCBI with the mass spectrometric data that obtains behind the K method enzymolysis BSA

Fig. 2 E searches the storehouse result for the NCBI that simplifies the mass spectrometric data that obtains behind the enzymatic isolation method enzymolysis BSA in the glue with the present invention

Embodiment

Method among the following embodiment if no special instructions, is conventional method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Be research material with salicornia europaeal Rubisco albumen and BSA among the following embodiment, enzyme solution (the Kumarathasan P that has systematically compared enzymatic isolation method (short cut technique) and latest report in the glue of simplification of protein of the present invention, Mohottalage S, Goegan P and Vincent R.An optimized protein in-gel digestmethod for reliable proteome characterization by MALDI-TOF-MS analysis.Analytical Biochemistry, 2005,346:85-89; Hereinafter to be referred as the K method) the mass spectrum identification result.

Used protein material salicornia europaeal Rubisco protein extraction is from the salicornia europaeal (Salicorniaeuropaea) of chamber planting, and the cultural method of salicornia europaeal is: behind the seed germination, with the pouring of 1/2Hoagland nutrient solution, per 3 days 1 time, irrigate.Draw materials after one month, with ℃ preservation of liquid nitrogen grinding postposition-80.

Bovine serum albumin (BSA) available from Sigma company (St.Louis, MO, USA).

Used reagent among the following embodiment:

(cat. no: Cat.No.11418033001), be the mass spectrum order-checking level product through modifying, the pure medicine of conventional analysis is available from the Beijing Chemical Plant available from Sweden Roche company for trypsase (Trypsin);

Used other reagent if no special instructions, all (Amersham Biosciences, Sweden), used water is ultrapure aqua sterilisa in dielectrophoresis and the mass spectrophotometry available from Amersham company.

When utilizing mass-spectrometric technique to identify albumen, embody the most key index of final qualification result quality and search storehouse score height exactly, score value is high more, illustrates that qualification result is reliable more, the confidence level of Identification of Fusion Protein is high more (RossignolM just, Peltier J B, Mock H P, Matros A, Maldonado A M, Jorr í n J V.Plant proteomeanalysis:A 2004-2006 update.Proteomics, 2006,6:5529-5548).In Mascot search, software is thought and is finally searched the storehouse score greater than 67 timesharing usually, and then confidence level reaches more than 95%, and the albumen of evaluation is believable.Embody Identification of Fusion Protein as a result the another one parameter of quality be the sequence coverage rate (SequencingCoverage, SC), the sequence coverage rate is high more, illustrate that the peptide section sequence of the target protein that comprises in the enzymolysis product is many more, qualification result is just reliable more certainly.In addition, the peptide section that obtained sum with can deserve the peptide section to get number also be important reference factor, peptide section sum is many more usually, the peptide hop count amount that can match is big more, the result is credible more.

Utilize These parameters to carry out compliance test result to enzyme solution in the simplification glue of the present invention below:

Enzyme solution enzymolysis salicornia europaeal Rubisco albumen and compliance test result thereof in embodiment 1, the simplification glue

In green plants, Rubisco albumen (rib μ lose-1,5-bisphospatecarboxylase/oxygenase) content is very abundant, can reach more than 50% of solubility total protein, and the Rubisco albumen of different plants has very high conservative property (Rossignol M, Peltier J B, Mock H P, Matros A, Maldonado A M, Jorr í n J V.Plant proteome analysis:A 2004-2006update.Proteomics, 2006,6:5529-5548).In mass spectrum research, detect the related experiment condition with Rubisco albumen usually and whether be fit to.

1, protein electrophorese and target protein point cuts

Take out method (Saravanan R S and Rose J C.A criticalevaluation of sample extraction techniques for enhanced proteomic analysisof recalcitrant plant tissues.Proteomics according to the improvement phenol of Saravanan and Rose, 2004,4:2522-2532) extract salicornia europaeal aerial part (shoot) total protein, press Bradford method quantitative protein (Bradford M M.A rapid andsensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding.Anal Biochem, 1976,72:248-254).Get 700 μ g salicornia europaeal aerial part total proteins, carry out bidimensional electrophoretic separation, the concrete operations of salicornia europaeal total protein dielectrophoresis are carried out according to Amersham company dielectrophoresis instructions.Dielectrophoresis (2-DE) result shows, in the salicornia europaeal aerial part total protein, and the big subunit content of Rubisco albumen equally very high (among Figure 1A shown in the arrow).Therefore, grope enzymolysis and carry out conditions such as mass spectrophotometry, database analysis with it.Carefully cut the abundantest protein site of content (Rubisco), be divided into 100 parts, then contain the 5 μ g Rubisco albumen of having an appointment in every part.Gel sample is placed 1.5 milliliters of clean centrifuge tubes, and-80 ℃ of preservations are standby.

2, simplify enzyme solution enzymolysis protein in the glue

The sample of a about 5 μ g Rubisco albumen in the gel that contains target protein point of-80 ℃ of preservations that step 1 is obtained, put the room temperature 10min that thaws, be divided into 5 equal portions, every part of protein content is 1 μ g Rubisco albumen (gel that contains this albumen is 50ug), get two parts, use enzyme solution and the interior enzyme solution of conventional glue in the simplification glue of the present invention respectively.Enzyme solution is according to people such as Kumarathasan ((Kumarathasan P in the conventional glue, Mohottalage S, Goegan P and Vincent R.An optimized protein in-gel digestmethod for reliable proteome characterization by MALDI-TOF-MS analysis.Analytical Biochemistry, 2005, method 346:85-89) is carried out (being called for short the K method).

The present invention simplifies enzyme solution in the glue, and the concrete operations step is as follows:

(1) washing: will have the gel (gel is 50ug) of 1 μ g Rubisco albumen to place 1.5 milliliters of clean centrifuge tubes, and add 100 μ l ultrapure water (ddH ₂O), mixing 30min gently, it is instantaneous that centrifugal (room temperature, 1000g 1min), abandon supernatant.Repeat this step once.

(2) decolouring: the washed gel of step (1), (pH is 8.5, and containing the quality percentage composition is that 50% acetonitrile (ACN) and quality percentage composition are 0.395%NH with 50 μ l destainers ₄HCO ₃Aqueous solution) decolouring twice, each 30min, during mixing 3 to 5 times.If color is taken off endless, can decolour again once, till the Coomassie brilliant blue color was taken off to the greatest extent, centrifugal then (room temperature, 1000g 1min), abandoned supernatant.

(3) dehydrate: in the centrifuge tube that the gel that step (2) decoloured is housed, add 50 μ l acetonitriles (ACN), put room temperature 5min, centrifugal (room temperature, 1000g abandon supernatant after 1min).Repeat once.Gentle breeze dries up about 1hr in super-clean bench, makes gel fully dry.

(4) preparation of enzyme working fluid: add 100mM hydrochloric acid to trypsase (Trypsin) dry powder (order-checking level product is modified by Roche company), the final concentration that makes enzyme is 0.1 μ g/ μ l, and normal temperature acidifying 10 minutes obtains Trypsin stoste.Be distributed into 20 μ l/ pipe ,-20 ℃ can store one month.The 50mM NH that in stoste, adds 9 times of volumes before using again ₄HCO ₃(pH 8.5), mixing promptly get Trypsin enzyme working fluid (the enzyme final concentration is 10ng/ μ 1).

(5) pre-enzymolysis: in the good gel of drying, add the Trypsin enzyme working fluid (10ng/ μ l) of 10 μ l steps (4) preparation, place 1h, enzyme liquid is fully immersed in the micelle for 4 ℃.Need not be centrifugal, inhale and remove unnecessary enzyme liquid.

(6) enzymolysis: add 8 μ l enzyme buffer liquid and (contain 1mM CaCL ₂With 25mM NH ₄HCO ₃, pH is 8.5 solution), this moment, the pH of enzyme digestion reaction system was 8.5,37 ℃ of water enzyme digestion 16hr.

(7) after enzymolysis was finished, the centrifugal 5min of 5000g carefully drew supernatant at normal temperatures, and supernatant is transferred in the PCR thin walled tube, directly utilized supernatant to carry out mass spectrophotometry.

3, the mass spectrum of enzymolysis product is identified and database search

The enzymolysis product that utilizes the substance assistant laser desorpted ionized time of-flight mass spectrometer (MALDI TOF MS/MS II) of Bruker company to carry out K method enzymolysis and simplify enzymatic isolation method is identified, and proofread and correct with matrix peak, the automatic degradation fragment of enzyme peak, measure the mass spectrometric data of each peptide section, concrete operations are carried out to specifications.

The result shows, utilizes to carry out MALDI TOF behind the K method enzymolysis Rubisco and analyze, and in the PMF collection of illustrative plates that obtains, 49 peptide sections arranged, karyoplasmic ratio (m/z) from 700 to 3000 (Figure 1B) that distribute; And carry out identical experiment with simplification enzymatic isolation method of the present invention, and can obtain 53 peptide sections, main peak mainly is distributed between 1000 to 2000, is beneficial to database search more, and the signal to noise ratio (S/N ratio) of main peaks (S/N) has also improved a lot (Fig. 1 C).These presentation of results utilize K method enzymolysis, can cause the peptide section to lose, and cause that mass-spectrogram part peak loses, thereby cause the Identification of Fusion Protein score to reduce, and with simplification enzymatic isolation method of the present invention, successful solution this problem.

To utilize mass spectrophotometry to obtain the PMF spectrum data and carry out data analysis by Mascot Distiller software, then by Matrixscience search website (http://www.matrixscience.com/search), (concrete querying condition: Enzyme:Trypsin under corresponding querying condition, Allow up to 1 missed cleavage, Peptide tolerance: ± 0.3Da, Mass values:MH ⁺, Monoisotopic:average Reporttop:20hits) searches for ncbi database, obtains the concrete authentication information of each albumen.The NCBI that simplifies the mass spectrometric data that obtains behind the enzymatic isolation method enzymolysis salicornia europaeal Rubisco albumen in the glue with K method and the present invention searches the storehouse result respectively shown in Fig. 1 D and Fig. 1 E.The result shows, the database search score height that short cut technique of the present invention obtains, and the Rubisco albumen that obtains with the K method must be divided into 104 fens (Fig. 1 D), and short cut technique must be divided into 117 fens (Fig. 1 E).

Enzyme solution enzymolysis bovine serum albumin (BSA) and compliance test result thereof in embodiment 2, the simplification glue

Bovine serum albumin (BSA) is the profuse a kind of albumen of content in the cow's serum, in ncbi database, has the lot of data information can be for reference, utilizes mass-spectrometric technique to identify the difficulty of this albumen thereby greatly reduce.For K method and simplification enzymatic isolation method are quantized comparison, be that experiment material has been carried out further comparative study with BSA.

Get 10 μ g-5ng BSA and carry out unidirectional electrophoretic separation, the BSA of different amounts separates back (Fig. 2 A) through unidirectional SDS-PAGE, cut protein band (Fig. 2 A that content is 0.1 microgram by hand, the band of arrow indication), be equally divided into 10 equal portions, then contain 10ng bovine serum albumin (weight that contains the gel of this albumen is 50ug) in every part of gel.Get two parts, simplify the interior enzymatic isolation method of glue with embodiment 1 described K method and the present invention respectively and carry out enzymolysis in the glue.

The enzymolysis product that utilizes the substance assistant laser desorpted ionized time of-flight mass spectrometer (MALDI TOF MS/MS II) of Bruker company to carry out K method enzymolysis and simplification enzymatic isolation method of the present invention is identified, and proofread and correct with matrix peak, the automatic degradation fragment of enzyme peak, measure the mass spectrometric data of each peptide section, concrete operations are carried out to specifications.Then according to same queries condition (concrete querying condition: Enzyme:Trypsin, Allow up to 1 missed cleavage, Peptide tolerance: ± 0.3Da, Mass values:MH ⁺, Monoisotopic:average Reporttop:20hits) carries out database search.Triplicate.The result shows, in the peptide finger-print (PMF) of MALDI TOF gained, utilizes K method enzymolysis can obtain 33 peptide sections (Fig. 2 B), and database search must be divided into 117 fens (Fig. 2 D); And can obtain 70 peptide sections with short cut technique, and quantity of information is about 2 times (Fig. 2 C) of K method, and Identification of Fusion Protein must be divided into 148 fens (Fig. 2 E), far away than the former height.These results further specify, and under the situation of protein content less (10ng), it is more obvious to simplify the enzyme solution superiority, can obtain more PMF data, provide more useful informations for searching the storehouse, thereby obtain higher score value, effectively improved the confidence level and the success ratio of identification of proteins.

Claims

1, a kind of method of protein gel internal enzymolysis comprises the steps:

3) adding contains Ca ²⁺Damping fluid, 37 ℃ of enzymolysis 14-18h remove gel and obtain protein enzymatic hydrolyzate.

2, method according to claim 1 is characterized in that: step 2) described in the concentration of hydrochloric acid be 100mM; Described NH ₄HCO ₃Concentration be 50mM; Enzyme concentration behind the described 100mM dissolving with hydrochloric acid trypsase is 0.08-0.12 μ g/ μ l; The trypsinase concentration of described trypsase working fluid is 8-12ng/ μ l.

3, method according to claim 2 is characterized in that: described step 2), the enzyme concentration behind the described 100mM dissolving with hydrochloric acid trypsase is 0.1 μ g/ μ l; The trypsinase concentration of described trypsase working fluid is 10ng/ μ l.

4, method according to claim 1 is characterized in that: in the described step 1), the consumption of ultrapure water is that 80-120 μ l/50 μ g contains protein gel, and wash time is 20-40min; Washing times is more than 2 times.

5, method according to claim 4 is characterized in that: in the described step 1), the consumption of ultrapure water is that 100 μ l/50 μ g contain protein gel, and wash time is 30min; Washing times is 2 times.

6, according to arbitrary described method among the claim 1-5, it is characterized in that: after also comprising washing in the described step 1), slough the step of Coomassie brilliant blue color in the gel with destainer; Described destainer is that 45-55% acetonitrile and quality percentage composition are 0.356-0.435%NH for containing the quality percentage composition ₄HCO ₃, the pH value is the aqueous solution of 8.0-9.0.

7, method according to claim 6 is characterized in that: described destainer is that 50% acetonitrile and quality percentage composition are 0.395%NH for containing the quality percentage composition ₄HCO ₃, the pH value is 8.5 aqueous solution.

8, according to arbitrary described method among the claim 1-5, it is characterized in that: soak dehydration with acetonitrile in the described step 1).

9, according to arbitrary described method among the claim 1-5, it is characterized in that: contain Ca in the described step 3) ²⁺Damping fluid for containing 0.8-1.2mM CaCl ₂With 20-30mM NH ₄HCO ₃, the pH value is the aqueous solution of 8.0-9.0.

10, method according to claim 9 is characterized in that: the described Ca of containing ²⁺Damping fluid for containing 1mMCaCl ₂With 25mM NH ₄HCO ₃, the pH value is 8.5 aqueous solution.