CN105277712A - Method for identifying single methylated modification of lysine epsilon-amino group side chain - Google Patents

Method for identifying single methylated modification of lysine epsilon-amino group side chain Download PDF

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CN105277712A
CN105277712A CN201410360062.3A CN201410360062A CN105277712A CN 105277712 A CN105277712 A CN 105277712A CN 201410360062 A CN201410360062 A CN 201410360062A CN 105277712 A CN105277712 A CN 105277712A
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lysine
monomethylation
cell
polypeptide
propionyl
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CN105277712B (en
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程仲毅
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Hangzhou Jingjie Biotechnology Co.,Ltd.
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PTM Biolabs Inc
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Abstract

The invention provides a method for identifying a lysine single methylated modification substrate. The method comprises the following steps: 1) an external acylated derivative reaction is used for deriving a single methylated epsilon-amino group of a substrate protein lysine residue to a propionyl methylated epsilon-amino group; 2) a affinitive enrichment is carried out for peptide fragments with propionyl methylated modification with a pan anti-propionyl methylated lysine antibody prepared by specificity; and 3) a mass spectrum identification is carried out for sites, polypeptide sequences and substrate proteins with propionyl methylated modification after affinitive enrichment by the antibody.

Description

A kind ofly identify the method that Lysine s-amino groups side chain monomethylation is modified
Technical field
The invention belongs to checkout and diagnosis field, especially, belong to lysine monomethylation in a kind of qualification and quantify cellular or tissue and modify the method for substrate; More particularly, belong to utilize the affine enrichment of specific antibody and the proteomics methodology of mass spectrophotometry is identified and quantify cellular or in organizing lysine monomethylation modify substrate.
Background technology
Single, double or tri-methylated modification can be there is in the epsilon-amino side chain of protein lysine residues.In the past few decades, mainly core histones is concentrated on to the methylate biological study of modification (Kme) of lysine.The research in early stage demonstrates this key effect be modified in chromosome structure and function enforcement.This decorating state had by 2 groups the enzyme of contrary catalysis regulate, i.e. lysine methylated transferase and lysine demethylase.Have at present and be found more than 50 lysine methylated transferases and about 25 lysine demethylases.Histone come propylhomoserin methylate modify with various disease association, such as cancer.Accordingly, the lysine regulatory enzyme that methylates becomes a series of potential drug target.
The current protein post-translational modification type (PTMs) found in histone all exists in nonhistones.Methylate in regulatory enzyme at the lysine identified, exist some acellular nuclear location enzyme and newfound be not that the lysine of substrate methylates regulatory enzyme with histone, illustrate that lysine methylates extensively distribute in nonhistones.Identification of protein substrate is the prerequisite determining protein post-translational modification function, and what lysine methylated that the importance of this prerequisite sets forth by biological research history is perfectly clear.Find after integrating we and other people data, lysine acetylation substrate appears at chromosome structure, transcriptional regulatory and as in metabolism three research fields, and has the plyability of substrate each other.Although the Substrate Identification that lysine acetylation is modified is comparatively slow, the research about it shows that lysine acetylation is synergy in above-mentioned three research fields.Similarly, the methylate qualification of modification group and quantitative comparison of lysine has found the nonhistones and signal path irrelevant with chromosome in some downstreams, and this is that follow-up functional study is laid a solid foundation.
Even so, lysine methylated substrate remarkable is identified.Introduce with qualification phosphorylation modification 32p marks difference, 3h or 14the low-activity of C makes radioactive isotope detection method be difficult to carry out in the qualification of lysine methylated substrate.Methylate, especially monomethylation (Kme1), in its substrate, only introduce a very little building stone, thus adorned sine group is trickle with the sine group difference not occurring to modify, only has a minimum conformation to be used for exploitation affinity antibody.To take into account compatibility and specificity simultaneously, the antibody that methylates (ubiquitin antibody) that exploitation has nothing to do with sequence is a huge challenge.Consequently, owing to not having the suitable peptide section enrichment method that methylates to provide peptide section for follow-up mass spectrophotometry, the qualification process of lysine methylated substrate is slow.Describe as in phosphorylation and the research of lysine acetylation group; affine enrichment is the committed step (Kim of Study on Protein posttranslational modification (PTM) holistic approach; S.C.etal.Substrateandfunctionaldiversityoflysineacetylat ionrevealedbyaproteomicssurvey.MolCell23,607-618 (2006)).The physico-chemical property difference of the lysine modified due to monomethylation and the lysine of unmodified is very little, therefore employing such as the chemical methodes such as the immobilized metal affinity chromatography (IMAC) of isolation of phosphorylated polypeptide are separated the very difficult (Ficarro of lysine polypeptide methylating and modify, S.B.etal.Phosphoproteomeanalysisbymassspectrometryandits applicationtoSaccharomycescerevisiae.Naturebiotechnology 20,301-305 (2002)).In addition, the antibody preparing the anti-lysine monomethylation of high specific and high-affinity is also very difficult.Therefore, need to carry out innovative ability based on proteomic techniques identify and analyze the lysine modification that methylates existing.Especially, need to lysine monomethylation modify Substrate Identification and quantitative analysis method carry out brand-new innovation and creation.
Summary of the invention
For solving this technical difficulty, this invention exploits a new chemical proteomics method.Utilize the method to identify efficiently, accurately in peptide section whether to occur the site that lysine ε-amido monomethylation is modified and modified, the change level that quantitative test is modified.
On the one hand, the invention provides a kind of identify in peptide substrate section or albumen whether occur on the ε-amido of lysine monomethylation modify method, method comprises: adopt external nitrogen acylation derivative reaction, allows the lysine residue of the monomethylation ε-amido on described substrate derive as acyl methylates ε-amido; With the methylated ubiquitin antibody of anti-acyl affine enrichment generation acyl methylate modify peptide section; Whether there occurs propionyl with the polypeptide of LC-MS mass spectrometer qualification antibody affine enrichment to methylate modification.
Preferably, detect the result obtained according to LC-MS mass spectrometer, and judge thus or estimate in substrate protein or peptide section whether in esse lysine list firstization is modified.
Preferably, 8 are less than to the total number of carbon atoms that substrate protein lysine residue monomethylation ε-amido carries out the modification group of nitrogen acylation modification.Preferred the total number of carbon atoms is less than 6.Preferably, this modification group can comprise alkyl or aromatic radical, and wherein the total number of carbon atoms of alkyl or aromatic radical is less than 8.Preferably, the total number of carbon atoms of alkyl is less than 6, or the total number of carbon atoms of aromatic radical is less than 8.
Preferably; the lysine residue of amido is allowed to derive as acetonyl ε-amido; butyryl methylates ε-amido; isobutyryl methylates ε-amido, and valeryl methylates ε-amido, and 2-methylbutyryl methylates ε-amido; 3-methylbutyryl methylates ε-amido; 2,2-dimethyl propylene acidylate ε-amido, methylate ε-amido or malonyl of succinylation methylates ε-amido etc.Acylating reagent comprises various carboxylic acid, acid anhydrides, acyl chlorides, and other all acylating reagents.
Preferably, acylating reagent is the acylating reagent with stable isotope.Isotope acylating reagent is carbon containing, hydrogen, oxygen, the carboxylic acid of nitrogen stable isotope, acid anhydrides, acyl chlorides, carboxylate, acid amides and other all acylating reagents.Preferentially, stable isotope reagent is 12c 6h 10o 3propionic andydride, 13c 6h 10o 3propionic andydride or 12c 6d 10o 3propionic andydride.
In other preferred modes, allow substrate protein or peptide carry out nitrogen acylation reaction and carry out in vitro.In some preferred modes, the acylation reaction of substrate protein or peptide can be carried out before protease digestion substrate or afterwards.Preferably, substrate protein or polypeptide are extracted from any cell, tissue or body fluid.Described substrate protein or polypeptide are total protein or total polypeptide.
In some preferred modes; from cell or tissue, extract total protein and propionyl acid anhydride is chemical reaction occurs in the hartshorn salt-sodium bicarbonate buffer liquid of 8.5 produce the propionating external derived protein of lysine in pH value, after reacting completely, produce the propionating derivative enzymolysis polypeptide of lysine through Trypsin Induced.In some preferred modes, the method preparing the propionating external derived protein of the propionating lysine reacted completely comprises: 200 μ L propionic andydrides are by the cell pyrolysis liquid that joins rapidly containing 20mg total protein, concussion also will add appropriate 2MNaOH pH will be adjusted to 8, then incubated at room 30 minutes; The propionating external derived protein of reacted lysine is by trichloroacetic acid (TCA) precipitation method precipitation.For producing the propionating derivative enzymolysis polypeptide of lysine further by the digestion damping fluid (0.1MNH of protein precipitation at 2ml 4hCO 3, pH=8.5) in resuspended, then with being dissolved in digestion damping fluid trypsase (trypsin) digestible protein 16 hours; After digestion completely, by 10 minutes centrifugal removing precipitations of 20,000xg, obtain the propionating derivative enzymolysis polypeptide supernatant of lysine.In preferred mode, the ratio of pancreatin and the propionating external derived protein of lysine is 1:100 (w/w).
In vitro in lysine derivative reaction; all can there is propionating reaction in ε-amido that unmodified lysine or monomethylation modify lysine; generate propionating lysine or propionyl to methylate lysine, and above-mentioned reaction can not be there is in di-methylation and tri-methylated lysine.React completely if propionating in theory, then reaction after albumen lysine residue on will there is not the lysine of unmodified.Therefore; reacted albumen is produced after enzymolysis polypeptide through enzymolysis; by mass spectral analyses lysine residue does not occur the polypeptide of any modification, compare the type polypeptide can assess propionating reaction reaction efficiency in the ratio of all lysine residue modified polypeptides identified.。
Some preferred embodiment in, described albumen (total protein or total polypeptide, or specific albumen or polypeptide), before carrying out propionating reaction, is extracted from cell culture medium cultured tumor cells or clinical sample.Preferably, cell is the tumour cell that can carry out going down to posterity based on the immortality of the cold labeling (SILAC) of cell chulture.Preferred, for lysine monomethylation in identification of cell modifies substrate, cell can be cultivated and be added with in the amino acid whose SILAC nutrient culture media of stable isotope.Preferentially, isotope amino acid comprises 12c-arginine or 13c-arginine, more preferentially, 12c-lysine or 13c-lysine, more preferentially, stable isotope amino acid is 12cH 3-methionine or 13cD 3-methionine.In some preferred modes, be the external methyl source providing lysine to methylate modification, with the addition of in cell culture medium 12cH 3-methionine ( 12cH 3-Met); Or, add in the medium 13cD 3-methionine ( 13cD 3-Met).Here, on methionine on Me 12c is by stable isotope 13c replaced, H 3(hydrogen) is by the D of stable isotope 3(deuterium) replaced.
Methylate in quantitative comparison a pair cell modify change time, need a cell chulture containing 12cH 3in the nutrient culture media of-methionine, another cell chulture is at cold labeling 13cD 3in the nutrient culture media of-methionine, the method is referred to as the amino acid cold labeling strategy (SILAC) (OngSEandMannM.Apracticalrecipeforstableisotopelabelingby aminoacidsincellculture (SILAC) .NatProtoc.1 (6): 2650-60. (2006)) based on cell chulture.
Some preferred embodiment in, the clone that described cell derived is bought in business, be selected from K562 cell (chronic myelogenous leukemia clone), SW620 cell (colon carcinoma cell line), one or more in A549 cell (Lines) and SMM7721 cell (hepatoma carcinoma cell).Tissue-derived for clinical diagnosis be the liver cancer tissue sample of liver cancer patient excision.
Some preferred embodiment in, need to carry out specificity anti-nitrogen acyl to methylate the affine enrichment of ubiquitin antibody to there is the methylate peptide section of modifying of nitrogen acyl.In preferred mode, the nitrogen acyl lysine ubiquitin antibody that methylates does not destroy in the matrix of Antibody avidity by chemical crosslinking, covalent bond or Non-covalent binding are any in advance.Described matrix is agarose resin, by Non-covalent binding on the agarose resin of albumin A or Protein G or albumin A/G; More preferentially, Non-covalent binding is prepared into antibody coupling resin on Protein-A Sepharose glucoresin.
Preferably, described ubiquitin antibody by chemical crosslinking, covalent bond or Non-covalent binding to not destroying in the matrix of Antibody avidity.Preferentially, by Non-covalent binding on the agarose resin of albumin A or Protein G or albumin A/G.More preferentially, Non-covalent binding is prepared into antibody coupling resin on Protein-A Sepharose glucoresin.
In some preferred modes, described antibody is the methylated ubiquitin antibody of specific bond propionyl.This enrichment method comprise the steps: general propionyl to methylate ubiquitin antibody and crosslinked agarose resin of albumin A carries out coupling, prepares protein A antibody binding resin.In some embodiments, affine enrichment is also included under 4 DEG C of conditions and propionyl is methylated ubiquitin antibody and the propionating derivative polypeptide night incubation of lysine.Or use antibody coupling resin and the propionating derivative polypeptide night incubation of lysine, then 1mlNETN damping fluid (100mMNaCl is used, 1mMEDTA, 20mMTrispH8.0and0.5% (w/v) NP-40) wash the antibody coupling resin after hatching; Then 0.1% (v/v) trifluoroacetic acid (TFA) of peptide section 100 μ l modified that methylated by the propionyl of enrichment affine in antibody resin washes through 3 wash-outs; Then eluent merges drains with concentrating instrument.
Some preferred embodiment in; before identifying adorned peptide section with LC-MS mass spectrometer; carry out HPLC separation to the peptide hydrolysis of albumen after carrying out propionating derivative reaction, the propionating derivative polypeptide fractions of the lysine after separation carries out propionyl respectively and to methylate the affine enrichment of ubiquitin antibody.Preferably, ionize process is carried out to the polypeptide of enrichment, then carries out the mass spectrophotometry of Nano-HPLC/MS/MS.Analyze through specific MASS SPECTRAL DATA ANALYSIS software (Mascot or Maxquant), identify specific propionyl on lysine residue to methylate and modify corresponding mass shift, and determine the albumen that monomethylation decorating site on polypeptide lysine residue, the quantitative change of modification, modified polypeptide sequence and this peptide sequence are corresponding thus.Finally obtain the result of lysine monomethylation being repaiied to the qualification of substrate and the quantitative change of feature site modification level.
Beneficial effect
By this method, accurately, specifically can identify and carry out the quantitative test of lysine monomethylation modification to lysine monomethylation modification in substrate protein.This is by method of the present invention, and we have authenticated to 460 monomethylation sites in 403 albumen, have very high degree of accuracy, obtains lysine monomethylation spectrum maximum up to now.There is fraction to be reported by former research institute in these 460 monomethylation sites, confirm the validity of the inventive method.And great majority are the site that lysine monomethylation unknown is in the past modified, thus greatly enrich the understanding that lysine monomethylation is modified.Lysine of the present invention methylate group analysis show that this is modified at the function in approach in born of the same parents, metabolism network and composite structure.Our result is that the functional study of follow-up lysine monomethylation path provides abundant data resource.
Accompanying drawing explanation
Fig. 1 is the experimental design process figure identifying lysine monomethylation polypeptide in the present invention's specific embodiment.Figure 1A is the experiment flow schematic diagram (lysine propionating external derivative reaction, antibody affine enrichment and mass spectrophotometry) of qualification containing monomethylation polypeptide; Figure 1B is conventional acylating reagent type; Fig. 1 C is for the experiment flow schematic diagram of the qualification of the propionating external derivative reaction of lysine, antibody is affine enrichment and mass spectrophotometry containing monomethylation polypeptide; Fig. 1 D is the chemical synthesising technology route map of the propionyl monomethylation lysine producing specificity propionyl monomethylation lysine ubiquitin antibody; Fig. 1 E is the specificity experiments that Dot blot (dot-spot) detects propionyl monomethylation antibody, wherein K pr+merepresent lysine propionyl methylated polypeptides storehouse, K proprepresent the propionating peptide library of lysine, K burepresent lysine Butyrylation peptide library, K merepresent lysine monomethylation peptide library, K me2represent lysine di-methylation peptide library, K me3represent the tri-methylated peptide library of lysine and K crrepresent lysine crotons acidylate peptide library.Fig. 1 F is a lysine propionyl methylated polypeptides from HSP90 albumen typical case's mass spectrophotometry annotated map.Pr:propionylation, propionating; Me:methylation, methylates; Pr+me:propionylmethylation, propionyl methylates; Bu:butyrylation, Butyrylation; Me2:di-methylation, di-methylation; Me3:tri-methylation, tri-methylated; Cr:crotonylation, crotons acidylate
Fig. 2 lysine external propionyl derivative reaction Efficiency testing.K562 cell holoprotein lysate is after the external propionyl derivative reaction of lysine, and trypsinization produces enzymolysis polypeptide, and mass spectrophotometry detects the propionating lysine in enzymolysis polypeptide.The propionating efficiency of lysine is by propionating lysine polypeptide the estimating of ratio in all polypeptide identified.
Fig. 3 lysine propionyl methylate decorative features spectrum.Fig. 3 A is warp 13cD 3the typical propionyl of-Met mark methylates the MS spectrogram of polypeptide modified.Fig. 3 B is the YSQADALK of EZH2 albumen in HeLa cell pro-methe MS/MS mass spectrogram of YVGIER polypeptide.Top is quilt 12cH 3the YSQADALK of-mark pr+meyVGIER peptide section MS/MS mass spectrogram, wherein, is below 13cD 3-mark the mass spectrogram of polypeptide, the b-of display same propionyl methylated polypeptides, the 4Da mass transfer of y-ion after cold labeling.Fig. 3 C be from " gently " ( 12cH 3-) " weight " ( 13cD 3-) the methylated polypeptides number comparison diagram that identifies in the HeLa lysis liquid mixture of methionine mark.The quantity of lysine monomethylation polypeptide that 20 polypeptide fractions Mass Spectrometric Identification after general propionyl methylates antibody richness that Fig. 3 D shows alkaline HPLC column separation acquisition arrives and the bioaccumulation efficiency of antibody.Pr:propionylation, propionating; Me:methylation, methylates; Pr+me:propionylmethylation, propionyl methylates.
The protein lysine monomethylation group of Fig. 4 high confidence level.Fig. 4 a is the Mascot ion score distribution plan of whole 463 the monomethylation polypeptide identified in all samples; Only show Mascot ion score higher than 30 polypeptide.Fig. 4 b is the Wien comparison diagram (HeLa (cervical cancer cell) of the lysine monomethylation polypeptide number identified from 5 tumor cell lines, K562 (chronic myeloid leukemia cells), SW620 (colon cancer cell), A549 (lung carcinoma cell) and SMM7721 (hepatoma carcinoma cell).
The mass spectrum proof diagram of propionyl methylation sites on CDC5L albumen K114 in Fig. 5 K562 cell.Fig. 5 a is the propionyl methylated polypeptides LANTQGK of synthesis pr+mek praK prr Tandem Mass Spectrometry Analysis, with from K562 cellular identification to identical polypeptides exhibit go out identical ionic strength figure, the top of figure is the polypeptide LANTQGK identified by the present invention in body pr+mek praK prthe mass spectrogram of R, below is the mass spectrogram of the polypeptide of synthesis.Fig. 5 b is polypeptide LANTQGK in body mek improvement on synthesis LANTQGK external with it methe Tandem Mass Spectrometry Analysis of K.Y-ion and b-ion part 4Da moves, and moves consistent with the position of lysine methyl residues.Pr:propionylation, propionating; Me:methylation, methylates; Pr+me:propionylmethylation, propionyl methylates.
The mass spectrum checking of Fig. 6 lysine propionyl methylated polypeptides.Mass spectrum annotated map comes from the contrast of lysine propionyl methylated polypeptides and corresponding improvement on synthesis in cell.Wherein, Fig. 6 A is KHDRSB1 Identification of Fusion Protein PVK in HeLa cell pr+me(the wherein K of GAYR peptide section pr+merepresent the lysine of propionyl monomethylation) second order ms (MS/MS) figure.Fig. 6 B is the mass spectrogram (PVK of corresponding external improvement on synthesis pr+megAYR).Fig. 6 C is the K of K562 cell EIF4H Identification of Fusion Protein pr+me(the wherein K of GGPDDR peptide section pr+merepresent the lysine of propionyl monomethylation) second order ms (MS/MS); Fig. 6 D is the second order ms figure (K of corresponding external improvement on synthesis pr+megGPDDR).Pr:propionylation, propionating; Me:methylation, methylates; Pr+me:propionylmethylation, propionyl methylates.
Fig. 7 is the modifier bit point diagram that the present invention identifies different cell, 5 tumor cell line (HeLa (cervical cancer cell), K562 (chronic myeloid leukemia cells), SW620 (colon cancer cell), A549 (lung carcinoma cell)) and SMM7721 (hepatoma carcinoma cell) in 29 lysine monomethylation modified proteins list of all identifying.
Describe in detail
the substrate mark that lysine monomethylation is modified
The amino acid whose modification that methylates is the abiogenous a kind of phenomenon of life entity or process.Show based on current research, in cell body, S-adenosine-L-Met (SAM) is the most important direct donor of methyl group, and first thiamine (Methionine, Met) is the direct prerequisite of synthesis SAM.Therefore, when adding methionine in the medium, after series reaction approach in cell body, the methyl group (CH on methionine 3-) ε-amido that will transfer to lysine forms the modification that methylates.Which constitute lysine methylate modify substrate mark General Principle, in cell culture medium, namely add the first thiamine of different cold labeling, as 12cH 3-Met or 13cD 3-Met, then will make on substrate protein leukorrhea respectively 12cH 3-methyl group or 13cD 3the methyl group of-Met.Mark approach is summarized as follows:
A: 12cH 3-methionine → S-adenosine-L methionine- -12cH 3(SAM) → lysine- 12cH 3(lysine monomethylation)
B: 13cD 3-methionine → S-adenosine-L methionine- -13cD 3(SAM) → lysine- 13cD 3(lysine monomethylation).
For the same sequences polypeptide modified containing a lysine monomethylation, adopt 12cH 3-Met or 13cD 3the sole difference of-Met mark is, due to stable isotope the difference of molecular weight, 13cD 3this polypeptide of-Met mark will compare 12cH 3the heavy 4.02Da of same sequences polypeptide of-Met mark (these two polypeptide differences a), thus can be come by the change of mass shift by Fig. 3 in mass spectrophotometry, and the amount of polypeptide itself number can be reflected by the abundance of mass spectrophotometry.Which constitute the theoretical foundation analyzing the protein modification quantitative test at different conditions of same albumen, be referred to as the amino acid cold labeling strategy (SILAC) (OngSEandMannM.Apracticalrecipeforstableisotopelabelingby aminoacidsincellculture (SILAC) .NatProtoc.1 (6): 2650-60. (2006)) based on cell chulture.Lysine monomethylation in cell or tissue is modified to the qualitative analysis of substrate, the labelling strategies of stable isotope need not be introduced, directly can be realized by the method for antibody enrichment, mass spectrophotometry.And for when in the consistent cell sample of quantitative test a pair genetic background, lysine monomethylation modifies level, then need to introduce 12cH 3-Met or 13cD 3the SILAC strategy of-Met mark, is realized by the method for antibody enrichment, mass spectrophotometry subsequently.
For how adopting mass-spectrometric technique to carry out qualitative or certain protein modification of quantitative test, comprising lysine and to methylate modification, the technical method that persons skilled in the art are known.
the propionating derivative reaction that lysine monomethylation is modified
Under normal circumstances, first the qualification of certain protein modification substrate needs to carry out affine enrichment, as the qualification of acetylation substrate with the specificity ubiquitin antibody of this modification to having this modified polypeptide.But lysine monomethylation is modified, due to the too small (CH of methyl group 3-) and very weak immunogenicity, be difficult to the ubiquitin antibody directly obtaining the modification of lysine monomethylation.In order to overcome this difficulty, inventor's imagination specificity can introduce extra group by external derivative reaction on lysine ε-amido that the modification of lysine monomethylation occurs, thus increasing the space structure of lysine ε-amido modification group, the preparation for antibody provides larger determinant and stronger immunogenicity.
The propionating reaction of lysine is the external derivative reaction that a kind of chemical reaction and synthesis skilled person know; by reacting under propionic andydride and substrate protein in vitro special reaction condition; on the lysine ε-amido of substrate protein, manually can introduce propionyl group, form the propionating lysine of derivatization.Modify for lysine monomethylation, owing to lysine ε-amido still having an extra propionyl anhydride reactant site, therefore also propionating reaction can occur, the propionyl producing derivatization methylates, and (Fig. 1 a) for lysine.And di-methylation is modified or ε-amido on tri-methylated modification lysine does not have can to produce propionyl derivative reaction (Garcia for propionyl anhydride reactant site, B.A.etal.Chemicalderivatizationofhistonesforfacilitateda nalysisbymassspectrometry.Natureprotocols2,933-938 (2007)) when lysine monomethylation modification in body methylates modification through external derivative reaction formation lysine propionyl, modification group greatly increases on space structure, more easily by antibody institute specific recognition.Therefore can prepare a species specificity in theory to know propionyl and to methylate the antibody of lysine, for identifying the lysine monomethylation modified polypeptide (Fig. 1 c) through propionating derivatization reaction.
First propionating for the lysine histone that is applied to (namely first passes through to extract histone by Garcia and Hunter two people; then lysine carries out to 5 kinds of histones after extracting propionating) modify (Garcia in research; B.A.etal.Chemicalderivatizationofhistonesforfacilitateda nalysisbymassspectrometry.Natureprotocols2; 933-938 (2007)) do not test this chemical method in more complex systems at that time, as whether the total protein in cell or tissue lysate is feasible.But first the present invention extracts the total protein of cell or tissue, and then externally carry out propionating reaction.After protease cracking, the method for antibody enrichment and mass spectrophotometry is adopted to analyze (detailed description sees below) lysine monomethylation group in holoprotein group aspect.
In a preferred mode, the K562 cell pyrolysis liquid holoprotein that the present invention uses and propionic andydride chemical reaction under pH value is the condition of 8.5.After reaction, after digesting in pH7.2 hartshorn salt-sodium bicarbonate buffer liquid with pancreatin, through the polypeptide of the propionating reaction of mass spectrophotometry generation lysine.If propionatingly to react completely external, the ratio containing unmodified lysine polypeptide will be very low.Qualification result shows; after propionating reaction; all detect 2; article 556, to only have in 37 polypeptide (accounting for 1.4%) containing the lysine of unmodified in polypeptide, illustrate that the external propionating reaction that adopts in the present invention has very high efficiency (Fig. 2) for the holoprotein system of complexity.
No matter be adopt 12cH 3-Met or 13cD 3-Met marks cell, mark 12cH 3-or 13cD 3-lysine monomethylation is modified all can there is propionating derivative reaction, generates propionyl 12cH 3-lysine or propionyl 13cD 3-lysine.Carrying out qualitative or quantitative test lysine monomethylation modification for by introducing propionating derivative reaction, adopting 13cD 3another necessary reason of-Met mark is, due to propionyl 12cH 3the mass shift that-lysine produces the same with lysine Butyrylation (70.0440Da), and propionyl 13cD 3-lysine but has the mass shift of unique 74.0640Da, thus the lysine Butyrylation can distinguished in mass spectrophotometry in body is modified, and reaches the effect that precise Identification lysine monomethylation is modified.
The enrichment of lysine propionyl methylated polypeptides and the preparation of ubiquitin antibody
Describe in phosphorylation and the research of lysine acetylation group, affine enrichment is the committed step that Study on Protein modifies holistic approach 11.The physico-chemical property difference of modifying lysine and unmodified lysine due to monomethylation is very little, therefore adopts the chemical method such as immobilized metal affinity chromatography (IMAC) as isolation of phosphorylated polypeptide to be separated the lysine polypeptide of the modification that methylates very difficult 15.In addition, the antibody preparing the lysine monomethylation of high specific and certain compatibility is also very difficult.For solving this difficulty, this invention exploits a kind of method of enrichment monomethylation polypeptide of novelty, the method comprises following three key steps: (1) react amino for the ε of monomethylation on lysine with propionic andydride, forms propionyl and to methylate ε amino chemical derivative; (2) the lysine antibody enrichment that methylates of specificity propionyl is utilized to methylate the polypeptide of lysine containing propionyl; (3) polypeptide of efficient liquid phase and mass spectrophotometry enrichment, identifies the sequence of antibody enrichment polypeptide and determines decorating site.
The propionating derivatization reaction of external introducing lysine is the prerequisite based on antibody enrichment method, and its principle and importance are at front detailed description.To methylate the ubiquitin antibody of lysine it is equally important that the inventive method needs to use specificity propionyl.Because propionyl methylated groups is than large, therefore prepares the methylate ubiquitin antibody of lysine of propionyl and prepare monomethylation lysine ubiquitin antibody frequently and want simple.For confirming that this is supposed, with immune rabbit after propionyl monomethylation lysine (Fig. 1 D) and KLH coupling, from rabbit anteserum, being purified into anti-propionyl subsequently methylating lysine ubiquitin antibody.Fixed position is utilized to contain propionating lysine (K pr), Butyrylation lysine (K buty), crotons acylated lysine (K cr), Dot blot (dotblot) method of the peptide library of monomethylation lysine (Kme1), di-methylation lysine (Kme2) and tri-methylated lysine (Kme3) measures antibody specificity (Fig. 1 c).Result shows, the propionating lysine ubiquitin antibody that methylates is stronger than other peptide libraries more than 100 times to the methylate specificity of lysine peptide library of propionyl.The structure of lysine and the similar of propionating lysine and Butyrylation lysine although propionyl methylates; but the propionyl that the testing result of Dot blot shows used by the present invention methylates, lysine ubiquitin antibody has very high compatibility and specificity, may be used for the enrichment of lysine propionyl methylated polypeptides.
One of main method of high throughput protein modification group based on the affine enrichment of antibody to modified polypeptide, as having a wide range of applications in lysine acetylation research.When the total protein extracted from cell or tissue is after external propionating reaction, can cut through proteinase enzyme and produce lysine propionating derivative polypeptide, comprise lysine propionyl and to methylate derivative polypeptide.In a preferred mode, proteinase is mainly trypsase (trypsin).Subsequently; methylate specificity propionyl lysine ubiquitin antibody and albumin A (ProteinA) agarose resin covalent coupling Dispersal risk binding resin; by antibody coupling resin in specific damping fluid with lysine propionating derivative polypeptide hatch; then have more the affine binding characteristic of specificity of Ag-Ab, the lysine propionyl derivative polypeptide that methylates can be affinely combined in antibody resin.After lavation buffer solution washes away non-specific binding polypeptide, elute with the elution buffer of the low ph value derivative polypeptide that the lysine propionyl of specific bond on antibody can be methylated.Carry out Nano-HPLC-MS/MS mass spectrophotometry after draining can identify lysine propionyl and to methylate derivative peptide sequence and determine decorating site.
propionyl monomethylation lysine polypeptide detects
Chemical modification group can make the mass shift of modification substrate and polypeptide, as-the CH that monomethyl is modified 2group can make substrate that the mass shift of 14.0147Da occurs, and phosphorylation modification can make substrate that the mass shift of 79.9663Da occurs.Lysine methylate occur mass shift the same with the mass shift that some amino acid mutations occur, if serine is for being mutated into threonine, aspartic acid is for being mutated into glutamic acid, and valine is for being mutated into leucine or isoleucine, and asparagine is for being mutated into glutamine.In addition, propionyl methylates and makes lysine that identical mass shift occur with Butyrylation.Lysine Butyrylation is also a kind of modification mode of body internal protein.Thus, when to whether certain Specific amino acid occurs monomethylation modify or external generation base group modification detects time, if do not have correct quality control, just probably there is false-positive result in qualification propionyl methylated polypeptides.Therefore, this law bright utilize existing 13cD 3the method of isotope labeling methylated polypeptides can be avoided occurring false-positive result.
Such as, when whether there is monomethylation modification on qualification lysine, during application isotope labelling method cultured cell, by the methionine in conventional medium 12cH 3replace to 13cD 3.In cell, known lysine methylates precursor-S-adenosylmethionine (SAM) from methionine conversion formation.Therefore, in cell, lysine methylates, 13cD 3methionine changes into 13cD 3s-adenosylmethionine ( 13cD 3-SAM), cause lysine that 18.027Da mass shift occurs. 13cD 3there is propionating rear generation 74.0640Da mass shift in vitro in the-lysine that methylates.And the mass shift that the mass shift of this uniqueness and other any known protein modification types or amino acid mutation occur can distinguish by Mass Spectrometer Method.As in one embodiment of the invention, a propionyl methylated polypeptides (Fig. 1 c) is identified from HSP90 albumen, by the mass shift of 74.0640Da, the 3rd lysine accurately can locating this polypeptide there is propionyl to methylate modification, and actually thus infer that this site there occurs lysine monomethylation and modifies.
For verifying the reliability of the method further, will 13cD 3methionine labeling method and SILAC methods combining.During experiment, HeLa cell parallelly cultivate is existed 12cH 3-methionine and 13cD 3in methionine nutrient culture media, Methyl groups is marked.After mark is complete, by two of identical amount kinds of crack proteins mixing, external propionating derivative reaction, then enzymolysis polypeptide is prepared in trypsinization.For reducing the complexity of sample and improving the flux of mass spectrophotometry, with preparative high performance liquid chromatography (HPLC) instrument isolated polypeptide, prepare 20 polypeptide fractions altogether.To each polypeptide fractions of preparation, carry out the affine enrichment of propionyl methylated polypeptides with the general propionyl lysine ubiquitin antibody that methylates, after enrichment polypeptide wash-out, nano-HPLC/MS/MS analyzes.
During mass spectrophotometry, occur lysine propionyl methylate the polypeptide modified because of hydrogen ion in methyl group with 12c carbon ion respectively replaced deuterium (D) ion with 13c carbon ion, will produce 4.002 daltonian mass shift (Fig. 3 A).For guaranteeing the accuracy analyzed, all MS/MS collection of illustrative plates searched for by MASS SPECTRAL DATA ANALYSIS software Mascot are further across artificial nucleus couple.Should in this way, the propionyl identifying 183 high confidence levels from HeLa cell methylates modified polypeptide (Fig. 3 C).In 20 enzymolysis polypeptide components, bioaccumulation efficiency is (Fig. 3 D) between 3% to 41%.By artificial nucleus to raw data, wherein, 180 propionyl modified polypeptide that methylates has a pair precursor ion, corresponding to 190 decorating sites that methylate in 162 kinds of albumen.Only have two polypeptide only containing " heavy (heavy) " ion, a polypeptide is only containing " light (light) " " ion.Explanation 13cD 3the method of replacing methyl group is used for monomethylation site atlas analysis and rejects false positive results being reliable.Also prove that this method is reliable for the identification of monomethyl modified polypeptide.
the scope of protein lysine monomethylation group
Then apply 13cD 3methionine labeling method have detected K562 cell (chronic myelogenous leukemia cell), SW620 cell (colon cancer cell), the lysine monomethylation in A549 cell (lung carcinoma cell) and SMM7721 cell (hepatoma carcinoma cell) 4 kinds of people source tumour cells is modified.Peptide identification adopt the false positive rate of strict 1% and reject MASCOT score value lower than 20 polypeptide, and the accuracy of each collection of illustrative plates of manual confirmation.After above-mentioned strict quality control, the present invention identifies 448 nonredundant lysine monomethylation modified polypeptides altogether in four tumour cells, 460 sites in corresponding 403 albumen.In these polypeptide, the Mascot score value of 73% is higher than 30 (Fig. 4 A).By retrieving public Uniprot database, qualification most of monomethyl site out is not yet reported.The monomethyl lysine polypeptide that the present invention identifies and the complete list of albumen are shown in Fig. 7.
The lysine monomethylation of core histones is modified and is extensively studied, and thus can be used for positive control as well.With estimate consistent, the present invention identifies 12 histone monomethyl decorating sites altogether.Except identify extensively research K4, K9, K27, K36, K79 of histone H 3 and the several methylation sites of K20 of histone H 4 except, also identify the K18 monomethylation decorating site of the histone H 3 of rare research.In 5 kinds of cells, there is these lysine monomethylation decorating sites due to known, the present invention has identified these sites and has illustrated that the method that this detection lysine monomethylation is modified is reliable and stable.Importantly, utilize method of the present invention, inventor also identifies some new decorating sites from much known monomethylation modified protein.As WIZ albumen, except identifying the lysine K976 site reported, also identify the lysine monomethylation site that K1321 and K1463 two kinds is new.
In order to verify the validity of the inventive method further, the lysine monomethyl that inventor then analyzes in the liver cancer tissue of people is modified, and studies the substrate spectrum that in clinical tissue, lysine monomethylation is modified.With identified lysine monomethylation in 5 kinds of tumour cells above and modify data and compare, result shows that 32 monomethylation sites in 29 monomethylation modified polypeptides in liver cancer tissue are all contained in these 5 kinds of cells, comprise the lysine monomethyl site (Fig. 7) of 20 new lysine methylation sites and 6 histone H 3s, show the reliability of the inventive method further
the checking of this case monomethylation decorating site of the bad ammonia of new qualification
The site-specific antibodie modified due to the lysine monomethylation that the overwhelming majority is nonhistones can not obtain in commercialization, thus the site of biochemical method to Mass Spectrometric Identification of Western blotting (westernblotting) cannot be utilized to verify.Therefore present invention employs the corresponding lysine monomethylation modified polypeptide of synthesis to the method for the lysine monomethylation modified polypeptide verified Mass Spectrometric Identification and go out.The modified polypeptide of lysine monomethylation polypeptide and corresponding Prof. Du Yucang in the cell that Mass Spectrometric Identification goes out, under identical mass spectrophotometry condition, should obtain identical second order ms figure (MS/MS), and this is the standard of phase homopolypeptide checking.For this reason, be the polypeptide between 20 to 60 from Mascot score value, pick arbitrarily propionyl monomethylation peptide sequence Prof. Du Yucang that 9 Mass Spectrometric Identifications go out.Methylate be modified to example, by the LANTQGK to Prof. Du Yucang CDC5L albumen K114 site in K562 cell to have identified propionyl pr+mek praK prthe mass spectrophotometry of polypeptide, after showing improvement on synthesis and the affine enrichment of antibody Mass Spectrometric Identification to polypeptide except the difference of 4Da caused because of isotope labeling, there is on all four second order ms figure (Fig. 5 A).Adopting except adopting except the method for improvement on synthesis verifies, in cell, also demonstrating the lysine monomethylation modification in CDC5L albumen.Mass spectrophotometry is carried out after being purified from the K562 cell cultivated by endogenous CDC5L albumen by immunoprecipitation.As previously mentioned, use 13cD 3-mark methyl group, then the CDC5L albumen of mass spectrophotometry purifying.In cell purifying CDC5L albumen identify the monomethylation polypeptide LANTQGK of K164 13cD 3-mek.For verifying this site further, by analyzing the collection of illustrative plates of corresponding improvement on synthesis, show with the collection of illustrative plates of polypeptide in born of the same parents except the difference of 4Da caused because of isotope labeling, basically identical (Fig. 5 B).
Same verification method also demonstrates the PVK of KHDRSB1 Identification of Fusion Protein in HeLa cell pr+methe second order ms figure (Fig. 6 A) of GAYR peptide section and the PVK of Prof. Du Yucang pr+methe second order ms figure (Fig. 6 A) of GAYR peptide section is consistent; At the K of K562 cell EIF4H Identification of Fusion Protein pr+mesecond order ms figure (Fig. 6 C) and the corresponding external improvement on synthesis K of GGPDDR peptide section pr+methe second order ms figure (Fig. 6 D) of GGPDDR is consistent.Demonstrate thus method provided by the present invention can high confidence level qualification lysine monomethylation modify.
Embodiment
The present invention adopts concrete embodiment to whether there is peptide that lysine monomethyl modifies in cell or tissue or albumen is identified or quantitatively, this enumerating is that how method of the present invention realizes in exemplary illustrating, can not play any restriction to the present invention.One of ordinary skill in the art can when doing any improvement and change without prejudice to when marrow of the present invention to the present invention, but this change is all comprised in the scope of claim of the present invention.
Examples of implementation 1: methionine cold labeling, external propionating derivatization reaction and proteolysis
1.1 methionine cold labelings
Cell used by the present invention, comprise HeLa cell (cervical cancer tumer line), K562 cell (chronic myelogenous leukemia cell), SW620 cell (colon cancer cell), A549 cell (lung carcinoma cell) and SMM7721 cell (hepatoma carcinoma cell), cultivate in RPMI or DMEM nutrient culture media (LifeTechnologies, CA) to carry out methionine cold labeling.following composition is mainly comprised in nutrient culture media: 12cH 3-methionine or 13cD 3-methionine (Sigma-Aldrich, MO), dialysis hyclone (v/v) (dialyzedFBS) (LifeTechnologies of 10%, the nutrient of CA) and 1 × microbiotic (ThermoScientific, MA) and other applicable Growth of Cells.In nutrient culture media, cell is at 37 DEG C and 5%CO 2in carry out grown cultures.
The key of cold labeling is that labeling effciency will reach more than 97%, and has Mass Spectrometer Method to be confirmed.A generation 50% is often divided according to cell 12cH 3-quilt 13cD 3-calculating (under physiological status be 12cH 3-), after cell divided for 6 generations continuously, more than 99% in body 12cH 3-quilt 13cD 3-replace, thus meet the cold labeling efficiency needed for theory." gently " carried out parallel ( 12cH 3-Met) or " weight " ( 13cD 3-Met) stable isotope cell marking process in, when " weight " ( 13cD 3-Met) after the mark of stable isotope reaches requirement, " gently " ( 12cH 3-Met) or " weight " ( 13cD 3-Met) cell of cold labeling needs to continue to cultivate until required cell quantity (10 8) enough albumen can be extracted for follow-up experiment.When cell chulture is to after meeting the demands, as carried out the Qualitative Identification that in cell, lysine monomethyl is modified, only collect " weight " ( 13cD 3-Met) carry out total protein extraction after the cell that marks.As carried out the quantitative test that lysine monomethyl in cell is modified, then " gently " ( 12cH 3-Met) or " weight " ( 13cD 3-Met) mixed in equal amounts carries out the extraction of albumen after the cell harvesting that marks.
the extraction of total protein in 1.2 labeled cells
The cell cultivating mark washes 3 times with the phosphate buffer (PBS) of precooling, then cell lysis (8M in the lysis buffer of precooling before collection ureabe dissolved in the 0.1MNH of 2:1 4hCO 3damping fluid (PH=8) and 0.1MNaHCO 3solution; 1x protease inhibitors (v/v)), finally hatch half an hour on ice.Centrifugal (20000xg) removes cell fragment, retains supernatant, and measures the content of total protein in supernatant fluid.
the external propionating derivatization reaction of 1.3 total proteins and proteolysis
The concrete step of the external propionating derivatization reaction of total protein is as follows:
1) get the Protein Extraction supernatant of each clone in 1.2 joints, wherein each supernatant contains 20mg total protein to prepare propionating reaction;
2) in the rich proteinaceous supernatant fluid of collection, add 200 μ L propionic andydrides immediately, vortex mixes, and adds the 2MNaOH of appropriate volume, pH is adjusted to 8.0, room temperature reaction 1 hour.
3) step 2 is repeated once.Rejoin in the solution of 200 μ L propionic andydrides in step 2, mixing, adds the 2MNaOH of appropriate volume, adjustment pH to 8.0, room temperature reaction 2 hours.
4) after reaction connects bundle, add 10 μ L monoethanolamines (ethanolamine) subsequently, room temperature 30 minutes, stop the propionating reaction of step 3.
5), in the solution in step 4, dropwise adding trichloroacetic acid (TCA) to final concentration is 20% (v/v), jog on shaking table, 4 DEG C of precipitates overnight.
6) second day 16,000Xg, 4 DEG C of centrifugal 10min, abandon supernatant.
7) the resuspended albumen precipitation of acetone of 4 DEG C of precoolings is added, then 16000Xg, 4 DEG C of centrifugal 10min, abandon supernatant.
8) step 7 twice is repeated.
9) 2mL100mM ammonium bicarbonate is added, the albumen precipitation in the resuspended step 8 of pH=8.
10) in the ratio of the substrate 1:50 (w/w) of pancreatin and albumen precipitation, add appropriate pancreatin in above-mentioned protein solution, in 37 DEG C of waters bath with thermostatic control, enzymolysis spends the night.
11) second day, centrifugal, adding dithiothreitol (DTT) (DTT) to final concentration is 5mM, reacts 30 minutes in 55 DEG C of waters bath with thermostatic control.
12) centrifugal, adding freshly prepared iodoacetamide aqueous solution to final concentration is 15mM, room temperature lucifuge reaction 30min.
13) after reaction terminates, centrifugal, add 0.72M halfcystine, to final concentration 30mM, room temperature reaction 30min.
14) again in the ratio of pancreas enzyme-to-substrate 1:100 (w/w), add appropriate pancreatin, 37 DEG C of water bath with thermostatic control enzymolysis are after 3 hours, 18the desalination of C post, by HPLC isolated polypeptide component.
examples of implementation 2: the polypeptide component based on HPLC is separated
Before affinity purification, the pancreatin peptide section of the propionating derivatization from each sample obtained by examples of implementation 1 is first separated by HPLC.The method be separated is as follows: this separation uses VarianSD1LC (AgilentTechnologies, CA) and XbridgeC18 (19X150cm; Waters, MA), adopt the bufferB (10mM ammoniacal liquor or 80%ACN, pH8.5) of 2 to 40%, gradient is set to 70 minutes, with flow velocity 10ml/min wash-out.Equal time collects sample, collects 60 components (about 10ml/ manages) altogether, then merges into 20 components.Each component of all 20 components uses Vacuum Concentration instrument (ThermoFisher) to drain respectively.
examples of implementation 3:lysine propionyl methylates the preparation of ubiquitin antibody binding resin and the affine enrichment of modified polypeptide
Lysine propionyl methylates the preparation of ubiquitin antibody binding resin: 1ml Protein-A Sepharose glucoresin (LifeTechnologies, CA) after the phosphate buffer (PBS) of precooling washs three times, and the 4mg lysine propionyl ubiquitin antibody (phosphate buffer in 2ml precooling) that methylates hatches 4 hours under 4 DEG C of conditions; After the centrifugal 30s of 500xg, the phosphate buffer of Antibody-protein A agarose resin precooling washs three times and removes unconjugated antibody, is prepared into lysine propionyl and methylates ubiquitin antibody binding resin.
The affine enrichment of modified polypeptide: the enzymolysis polypeptide (2mg) obtained by embodiment 2 is dissolved in the NETN damping fluid (100mMNaCl of 200ul precooling, 1mMEDTA, 20mMTrispH8.0and0.5% (w/v) NP-40), then add 20ul lysine propionyl to methylate ubiquitin antibody binding resin, night incubation under 4 DEG C of conditions.The centrifugal 30s of 500xg, after affine enrichment peptide-antibody-Protein-A Sepharose glucoresin NETN buffer solution three times, then washes twice with ddH2O.Be combined in 0.1% trifluoroacetic acid (TFA) wash-out of the affine enrichment polypeptide 100ul on Antibody-protein A agarose resin, totally three times.Mix the eluent of three times, for follow-up mass spectrophotometry after draining with Vacuum Concentration instrument.
Examples of implementation 4:HPLC-MS/MS analyzes and database retrieval protein sequence
The ionization of wash-out peptide section: the peptide section that methylated by the propionyl be enriched in examples of implementation 3 (the peptide sections of 20 components) is dissolved in 3 μ lHPLC buffer A (0.1% aqueous formic acids respectively, v/v) kapillary RPLC trapping column (100 μm of internal diameter x2cm are then entered successively, LunaC18 fills, 5 μm, aperture china enlightening horse) and maximum pressure be the self-actuated sampler of 250 handkerchiefs, damping fluid is 100% buffer A (the 0.1%FA aqueous solution of HPLC level).After sample introduction and wash-out terminate, peptide section is transferred to fill C18 resin (3-μm of grain size, aperture, Chinese enlightening horse) analytical column (10cm is long, 75 μm of ID), and analytical column is connected EASY-nLC1000HPLC system (ThermoFisherScientificInc, MA).
Mass spectrophotometry: after the peptide section eluted is ionized, then imported in LTQOrbitrapElite mass spectrometer (ThermoFisherScientificInc, MA) by nano-spray.With R=24,000 and the resolution of m/z400, carry out gamut scanning of the mass spectrum (from m/z300 to m/z2,000).10 kinds of ions the most abundant are once separated in linear ion hydrazine, and then divide (CID) through collision, this normalized energy is 35%.Be spaced apart 30 seconds with the eliminating that data are irrelevant, be recounted as 2, get rid of window and be arranged on+2Da and-1Da.The HPLC-MS/MS data acquisition obtained is analyzed with Mascot (v2.3, MatrixScience, UK).Peak list is by the extract_msn.exe Software Create of ThermoFisher.During Mascot analyzes, parent ion quality error is set to ± 10ppm, and fragment masses error is set to ± 0.6Da.The data obtained is searched in UniProtHuman (88,817 sequence) database, and search parameter is done following setting: fixing modification is set to cysteine residues urea and methylates, variable being modified to of inferior quality peptide section 13cD 3-methionine, 13cD 3-methionine oxidation, lysine propionating (lysine+56.0262), lysine propionyl 13cD 3-to methylate (lysine+74.0640) and lysine propionyl-methyl-methylate (lysine+70.0418).All mass spectrometric datas adopt the mascot ionic fraction higher than 20 to filter and manual verification.
The purifying of CDCL1 albumen in examples of implementation 5:K562 cell
Will 13cD 3the heavy target K562 cytolysis of-methionine is in 1xNETN damping fluid (100mMNaCl, 1mMEDTA, 20mMTrispH8.0 and 0.5%w/v, NP-40).Cell homogenates is 12, and 000 × g, goes out cell fragment under 4 DEG C of conditions centrifugal 10 minutes.
Add anti-CDC5L antibody (SantaCruz, Texas) 4 DEG C in lysate to spend the night, then add albumin A/G agarose resin (SantaCruz, Texas) and hatch 4 hours at 4 DEG C.After washing non-specific binding albumen, immunoprecipitate is above separated at 4 – 12%SDS-PAGE (LifeTechnologies, CA) after thermal cracking in sample-loading buffer.Adhesive tape corresponding for destination protein is cut and at film dosim, then peptide hydrolysis is dissolved in 0.1%TFA damping fluid and imports in the HPLC-MS/MS mass spectrum of examples of implementation 4 and analyze.
Examples of implementation 6: the preparation of total protein in hepatoma sample
Liver cancer tissue comes from and carries out the liver cancer patient of performing the operation in Zhong Shan hospital (China, Shanghai), and research contents has been informed with patient or contributed people.The flesh tissue cut before doing further process by liquid nitrogen quick freeze.Before extracting albumen, liver organization uses rapidly surgical scissors to shred and uses the phosphate buffer of precooling (PBS) to wash away blood.The tissue being placed in the PBS of precooling uses stirrer to stir, and connective tissue is with filtrator filtering (70 μMs of apertures).By centrifugal acquisition hepatoma carcinoma cell, and carry out cell chulture and mark by the method for 1.1 joints in embodiment 1, cell culture medium carries out cultivate and use 12cH 2-methionine or 13cD 2-methionine marks.Carry out the extraction of total protein through the method for 1.2 of examples of implementation 1 and 1.3 joints and carry out propionating reaction after cell marking; also decompose according to identical method enzyme simultaneously; adopt the degree of the propionating reaction of chromatography analysis; result shows; in the protein of these cells; only have few peptide not carry out propionating reaction, all albumen of essence has all carried out propionating reaction.
Examples of implementation 7: for the identification of the preparation of the methylated ubiquitin antibody of the third lysine propionyl
The following list of the reagent used in the embodiment of the present invention:
Table 1
In the embodiment of the present invention, various solution formula is as follows:
Table 2
The preparation of ubiquitin antibody
The synthesis of 1, the micromolecular synthesis of the propionating lysine of monomethyl (Lys (me-prop)-OH).
Synthetic route (Fig. 1 D, wherein NHFMor is blocking group):
1) 2-1 methylene chloride (DCM) dissolves, and continues to pass into brand-new HCl gas after 1.5 hours under normal temperature, and under strong acid, normal temperature continues reaction 5 hours, and TLC confirms that reaction terminates; After being spin-dried for sticky solid, then add silica gel mixed sample and carry out column chromatography and obtain 2-2;
2) 2-2 adds acetone solution, is adding NaHCO 3after aqueous solution, stirring at room temperature 2 hours (fully stirring, the HCl combined in removing raw material), slowly adds the acetone soln being dissolved with propionic andydride, reacts 1 hour under ice-water bath; Reaction terminates rear 2N hydrochloric acid and adjusts about pH3.0, adds DCM and extracts, get organic phase and be spin-dried for obtain sticky solid, adds silica gel and carries out column chromatography and obtain 2-3;
3) 2-3 adds piperidines after adding DMF (DMF) and tetrahydrofuran (THF) dissolving, and stirring at normal temperature is spent the night, and TLC confirms that reaction terminates; Oil pump obtains mix products after subtracting steaming solvent evaporated.Add purification by silica gel column chromatography and obtain esterification products; Hydro-oxidation sodium, etc. water gaging and methanol system be hydrolyzed, react 2 hours under ice-water bath, after esterification products complete hydrolysis, be adjusted to pH3.0 with 2NHCl.Be spin-dried for solvent, then add ethanol lysate, after filtering, filtrate is spin-dried for, and obtains final product Lys (me-prop)-OH.
2, the modified lysine Small molecular holoantigen preparation of activation
1) appropriate KLH is dissolved in 1mL activation buffer, adds the EDC of 3.2mg, makes EDC final concentration 2mM;
2) 1.1mgSulfo-NHS is added in reactant liquor 1;
3) fully reactant liquor 1 and 2 is mixed, room temperature reaction 15 minutes;
4) add 1.4 μ L mercaptoethanols and stop EDC reaction;
5) appropriate Lys (me-prop) is dissolved in activation buffer, adds in the KLH protein solution after activation subsequently, adjustment pH to 7.4;
6) abundant mixed reaction solution, reacts 2 hours under room temperature;
7) 50mMTris cessation reaction is added.
3, animal immune program
1) 8 week age new zealand white rabbit, subcutaneous multi-point injection;
2) first time immunity: getting 500 μ L concentration is that the small molecule immune of the KLH coupling of 0.8mg/ml is former in same volume Split completely mixing and emulsifying, the immunity of dorsal sc multiple spot;
3) after 3 weeks, after getting the immunogene and the incomplete freund adjuvant mixing and emulsifying of same volume that 500 μ L concentration are 0.4mg/ml, dorsal sc multi-point injection;
4) every 2 weeks, by the dosage of step 3 and method booster immunization once;
5) the 4th immunity gets 1ml serum in latter 10 days, detects serum titer by ELISA method.If serum titer is greater than 30,000, namely pass through Culling heart blood.If serum titer is defective, then continue to repeat step 4, until serum titer is greater than 30,000.
4, polyclonal antibody purification
ProteinA prepurification:
1) titre centrifugal 10 minutes of serum 10000r/min being greater than 30,000, draws supernatant 0.45 μm of filtering with microporous membrane;
2) 3mlProteinA resin equilibrium at room temperature 1 hour, the phosphate buffer (PBS) of 4 times of column volumes cleans resin;
3) filter after serum 20ml be loaded in the pillar containing ProteinA resin, and with ProteinA pillar incubated at room 90min after, leave standstill 15min;
4) wash 1 time with the cleaning buffer solution A of 10 times of column volumes respectively, 15 times of column volume PBS wash pillar 1 time;
5) the wash-out Buffer wash-out of 5 times of column volumes, adds neutralization buffer subsequently, 4 DEG C of PBS dialysed overnight;
6) ultrafiltration IgG is 5-10mg/ml to concentration.
Prepared by antigen polypeptide coupling pillar
1) equilibrium at room temperature SulfoLinkCouplingResin1 hour, draws 5ml in chromatography void column after mixing, the coupling buffer cleaning of 4 times of column volumes twice;
2) take 5mg antigen polypeptide, be dissolved in coupling buffer respectively, add in SulfoLinkCouplingResin chromatographic column post and mix, incubated at room 15 minutes;
3) leave standstill 30 minutes, the coupling buffer of 15 times of column volumes washes pillar;
4) add 1 times of column volume Block buffer, incubated at room is after 15 minutes, and 6 times of column volume cleaning buffer solution B clean pillar;
5) obtain respectively containing polypeptide 1 coupling pillar and polypeptide 2 coupling pillar.
Antigen polypeptide affinity purification
IgG after ProteinA purification ultrafiltration is added in antigen polypeptide coupling pillar, incubated at room 2 hours; After giving up efflux, clean pillar with the PBS of the cleaning buffer solution of 5 times of column volumes and 10 times of column volumes respectively, then movable antibody purification according to the following steps:
1) 4 times of column volume elution buffer wash-outs;
2) neutralization buffer is added in eluent, in 4 DEG C of PBS dialysed overnight in bag filter;
3) ultrafiltration IgG is 5-10mg/ml to concentration;
4) IgG after above-mentioned ultrafiltration is added in polypeptide 2 coupling pillar, incubated at room 30 minutes, collects efflux, be the antibody that purifying is good.
5, Dot blot (DotBlot) detects:
1) by each peptide species (K pr+merepresent lysine propionyl methylated polypeptides storehouse, K proprepresent the propionating peptide library of lysine, K burepresent lysine Butyrylation peptide library, K merepresent lysine monomethylation peptide library, K me2represent lysine di-methylation peptide library, K me3represent the tri-methylated peptide library of lysine and K crrepresent lysine crotons acidylate peptide library) soluble in water, be configured to the polypeptide solution that initial concentration is 100ng/ μ L;
2) pvdf membrane of the suitable size of cutting, about 40 seconds of absolute methanol process, rinsing 3 times in deionized water, natural drying 2 minutes;
3) by initial concentration be 100ng/ μ L polypeptide solution further respectively dilution for 20ng/ μ L and 4ng/ μ L.Then point sample successively, 1 μ l/ point, puts into six orifice plates after dry 10 minutes;
4) 5% skimmed milk power room temperature closes 60 minutes, TBST washing lotion rinsing twice, 5 minutes/time;
5) with 5% skimmed milk power dilution antibody, incubated at room 1 hour, with TBST washing lotion rinsing three times, 10 minutes/time;
6) with 5% skimmed milk power dilution goat-anti rabbit HRP labelled antibody, room temperature 45 minutes, TBST washing lotion rinsing three times, 10 minutes/time;
7) chemiluminescence nitrite ion is evenly laid on film, hatches post-exposure in 5 minutes, the results are shown in Figure 1E.
Dot blot detects and shows; the polyclonal antibody that the monomethylation designed by the present invention propionating lysine Small molecular is obtained as antigen can specific recognition lysine methyl-prop acyl polypeptide storehouse, lysine methyl-prop acidylate GG polypeptide, monomethylation propionating lysine Small molecular and KLH coupling the propionating lysine Small molecular of monomethylation; but also the polypeptide (Fig. 1 E) of nonrecognition other types polylysine modification, describes the specificity that antibody is good thus.The propionating lysine polyclonal antibody of the monomethylation developed can identify the lysine methyl-prop acyl polypeptide storehouse of 4 nanograms; but nonrecognition similar until other modified lysine peptide libraries of 100 nanograms; this had both shown the sensitivity of developed antibody, also indicated the specificity of this polyclonal antibody further.
as a result 1
For HeLa cell, according to the method for above-mentioned fact Example 1-4 identify in HeLa clone whether occur lysine monomethylation modify and occur modify site, need by 13cD 3methionine labeling method and SILAC methods combining.During laboratory, by HeLa clone respectively parallelly cultivate in containing 12cH 3-methionine and 13cD 3in methionine nutrient culture media.Cell marking is completely and when reaching the amount of needs; collecting cell extracts total protein respectively; then carry out external propionating derivative reaction by after two of identical amount kinds of crack protein mixing, trypsinization produces enzymolysis polypeptide, produces 20 polypeptide fractions with preparation HPLC isolated polypeptide.Each the polypeptide fractions lysine propionyl be separated methylates ubiquitin antibody enrichment, then analyzes (specific analytical method is shown in examples of implementation 4) with nano-HPLC/MS/MS.
During mass spectrophotometry, occur propionyl methylate modify polypeptide because of hydrogen ion in methyl group and carbon ion all replaced, 4.002 daltonian mass shift (Fig. 3 A) will be produced.For guaranteeing the accuracy analyzed, all MS/MS collection of illustrative plates searched for by Mascot are further across artificial nucleus couple.In this way, the hydroxypropyl methyl modified polypeptide of 183 high confidence levels should be identified from HeLa cell, thus indirectly demonstrate the existence that there is the lysine that 183 monomethylations are modified in HeLa cell.By artificial nucleus to raw data, wherein, 180 hydroxypropyl methyl modified polypeptides have a pair precursor ion, corresponding to 190 decorating sites that methylate (Fig. 3 C) in 162 kinds of albumen.Only have two polypeptide only containing " heavy (heavy) " ion, a polypeptide is only containing " light (light) ion.
This absolutely proves 13cD 3the method of replacing methyl group is used for monomethylation site atlas analysis and rejects false positive results being reliable.Also prove that this method is reliable for the identification of monomethyl modified polypeptide.
as a result 2
For application 13cD 3methionine labeling method have detected K562 cell (chronic myelogenous leukemia cell), SW620 cell (colon cancer cell), A549 cell (lung carcinoma cell) and SMM7721 cell (hepatoma carcinoma cell)) etc. the lysine monomethylation in other 4 kinds of people source tumour cells modify, the step of concrete steps described by examples of implementation 1-4 and condition.Wherein, peptide identification adopts the false positive rate of MASCOT algorithm and 1%, rejects MASCOT score value lower than the record of 20, adopts strict standard, each collection of illustrative plates of manual confirmation (as previously mentioned) 22.Altogether identify 448 nonredundant monomethyl polypeptide, 460 sites in corresponding 403 albumen.In the polypeptide that these identify, the MASCOT score value of 73% is higher than 30 (Fig. 4 A).By retrieving public Uniprot Protein Data Bank, the present invention the not yet open report in most of monomethyl site of identifying out.
The monomethyl lysine polypeptide identified by the present invention and the complete list of albumen are seen attached list.This shows, use method of the present invention can go out those monomethyl lysine polypeptide identified by additive method and albumen by precise Identification, meanwhile, also identified a lot of before other monomethyl lysine polypeptide occurred and the albumen that can not identify.Fig. 7 shows 29 lysine monomethylation modifying proteins all identifying in five tumor cell lines and decorating site.
From the result of above qualification, use method of the present invention, the polylysine modification that before can identifying those, ignorant monomethylation is modified.Such as on the protein substrate of STUB1 gene code, the present invention detects the existence that lysine monomethylation is modified on the lazy propylhomoserin (K2) of the 2nd position, and the modification of the lysine monomethylation in this site is not yet in the news.
The lysine monomethylation of core histones is extensively studied, and thus can be used for positive control as well.With estimate consistent, the inventive method identifies the lysine monomethyl site of 12 core histones altogether.Except identifying the histone H 3 K4 of extensively research, outside H3K9, H3K27, H3K36, H3K79 and the several methylation sites of histone H 4 K20, also identify the histone H 3 K18 monomethylation site of almost no research.Owing to all containing these monomethylation decorating sites of major part in these five kinds of cells, illustrate that the method that this detection lysine monomethylation is modified is reliable and stable.In addition, the inventive method also identifies some new decorating sites from much known monomethylation modified protein.As in WIZ albumen, except identifying the K976 site reported, the present invention also identifies the new monomethylation site (Fig. 7) of K1321 and K1463 two kinds.
as a result 3
In order to verify the reliability of the inventive method, with 13cD 3the K562 cell of-Met mark is example, by method of the present invention, identifies and there is monomethylation modification on the K114 site of the CDC5L albumen of K562 cell.The total protein of K562 cell is after enzyme of going forward side by side through external propionating reaction is cut, and the sequence of a polypeptide of acquisition is LANTQGK * pr+mek praK prr (K * pr+merepresent propionating monomethylation lysine, K prrepresent and be also connected with propionyl group on lysine).Can know from description above, before this peptide sequence does not carry out external propionating reaction, this peptide sequence should be in biological cell or tissue: LANTQGK me *kAKR (K *represent in this site, namely 114 sites there occurs monomethylation and modify).In order to verify this conclusion, inventor is to the LANTQGK of Prof. Du Yucang * pr+mek praK prr has carried out Tandem Mass Spectrometry Analysis.Result shows, this improvement on synthesis and the LANTQGK identified from cell * pr+mek praK prr polypeptide has duplicate MS/MS secondary spectrogram, but on corresponding b-, y-ion, have the mass shift (Fig. 5 A) of 4Da.In order to verify that this site there occurs lysine monomethylation truly and modifies in cell body further, identify that lysine monomethylation modifies with Tandem Mass Spectrometry Analysis that is artificial synthetic polypeptide after the present invention additionally uses cell marking in body after CDC5L protein purification.Identify in the CDC5L albumen of purifying polypeptide.To Prof. Du Yucang the mass spectrophotometry of polypeptide shows, the MS/MS secondary spectrogram of this polypeptide and purifying CDC5L Identification of Fusion Protein arrive unanimously, only because of 13cD 3-be marked at the mass shift corresponding b-, y-ion having 4Da, demonstrate the reliability that the lysine monomethylation that identifies in CDC5L albumen is modified thus.
The qualification of the lysine monomethyl modification in the liver cancer tissue of people is demonstrated further to the reliability of the inventive method.The data of modifying atural object with the lysine monomethylation identified in above-mentioned five kinds of cells compare, 32 monomethylation sites in identify 29 monomethylation modified polypeptides in liver cancer tissue are all contained in these 5 kinds of cells, comprise the lysine monomethylation decorating site (Fig. 4 B, Fig. 7) of 20 new methylation sites and 6 histone H 3s.
Discuss
Although the protein science strategy based on affine enrichment has been used to many peptide sections that methylate of identification of cell lysate recently, still there is challenge, particularly monomethylation is modified the small physicochemical property caused and is made it be difficult to identify by the method for conventional lysine monomethylation antibody enrichment.Utilize method of the present invention, inventor identifies 460 lysine monomethylation sites in 403 substrate proteins, learns data for maximum group that this represent current lysine monomethylation.This result clearly confirms the validity and reliability of method of the present invention.
Although very effective for lysine monomethylation, our method is not also suitable for lysine di-methylation and tri-methylated group horizontal detection because the amino acid residue of these two kinds modifications can not with propionyl anhydride reactant.We also use 13cD 3-methionine labelling strategies improves the degree of accuracy detecting monomethylation peptide section.But this method can not directly apply to clinical tissue sample, the reliability of polypeptide identification also can alternative method solve, and the MS/MS of such as strict manual verification and improvement on synthesis analyzes.In recent years, the biological relevance of protein lysine monomethylation causes the extensive concern of research institution.Protein lysine methylates enzyme may target spot by the medicine as various disease.In addition, the inhibitor of some lysine Regulation by Methylation enzymes is at present at clinical assessment.Such as, but the existing knowledge of lysine monomethylation is confined to a limited number of albumen, histone and p53, thus limit the application that lysine monomethylation is modified at basic medical research and even course of drug development.Our research provide not only a kind of method of effective lysine monomethylation substrate high throughput identification, and extends the known directory of lysine monomethylation albumen.The genetic manipulation that proteomics method described in the invention and the enzyme that methylates are expressed is combined, can apply to identify the performance analysis of methylated substrate under the substrate of transmethylase and demethyl enzyme, disease conditions.In addition, lysine monomethylation modification group provided by the present invention will be the meaningful starting point of further biological study, and the functional characterization realizing lysine monomethylation albumen describes the parsing with disease specific lysine monomethylation approach.

Claims (21)

1. ε-the amido identifying lysine residue in substrate there is the method that monomethylation is modified, the method comprises: 1) adopt external nitrogen acylation derivative reaction, is derived by the lysine residue monomethylation ε-amido on substrate as acyl methylates ε-amido; 2) acyl utilizing specificity to prepare methylate the affine enrichment acyl of lysine ubiquitin antibody methylate modify peptide section; 3) acyl of mass spectrum and data analysis qualification antibody affine enrichment is utilized to methylate the site and polypeptide modified.
2. method according to claim 1, substrate lysine residue monomethylation ε-amido derives as propionyl methylates ε-amido.
3. method according to claim 2, utilize propionyl methylate lysine ubiquitin antibody to propionyl methylate modify site carry out enrichment.
4. method according to claim 1, carry out nitrogen acylation modification to substrate lysine residue monomethylation ε-amido, the total number of carbon atoms of modification group is less than 8, and preferably, modification group comprises alkyl or the aromatic radical that the total number of carbon atoms is less than 8.
5. according to the method one of claim 1-4 Suo Shu, described substrate is polypeptide or albumen, and acylation reaction can be carried out before protease digestion substrate or afterwards.
6. the method according to claim 4 or 5, utilize corresponding substrate protein acyl methylate lysine ubiquitin antibody to acyl methylate modify site carry out enrichment.
7. the method according to claim 1-6, the substrate that the lysine residue that identify ε-amido generation monomethylation is modified is be extracted from the albumen or polypeptide that obtain in any cell, tissue or body fluid.
8., according to the method one of claim 1-7 Suo Shu, the substrate protein white matter being extracted from any cell, tissue or body fluid can carry out external chemical derivatization reaction; Acylating reagent is the acylating reagent with stable isotope; Preferentially, stable isotope reagent is 12c 6h 10o 3propionic andydride, 13c 6h 10o 3propionic andydride and 12c 6d 10o 3propionic andydride.
9. method according to claim 8, isotope acylating reagent is carbon containing, hydrogen, oxygen, the carboxylic acid of nitrogen stable isotope, acid anhydrides, acyl chlorides, carboxylate, acid amides and other all acylating reagents.
10. method according to claim 8, modifies substrate cell to lysine monomethylation in cell and carries out external propionating reaction, both can carry out the modification of lysine monomethylation Qualitative Identification ( 12c 6h 10o 3mark), also can carry out the modification of lysine monomethylation quantitative test ( 12c 6h 10o 3, 13c 6h 10o 3or 12c 6d 10o 3mark).
11. methods according to claim 10, modify the quantitative test of substrate to lysine monomethylation, the analysis of stable isotope acylating reagent can be selected more to organize lysine monomethylation and modify substrate; Analyze three groups of different modification substrates that methylate, can carry out 12c 6h 10o 3, 13c 6h 10o 3with 12c 6d 10o 3mark respectively.
12. according to one of claim 1-11 or described method, and cell is the cell that can carry out going down to posterity based on the immortality of the cold labeling (SILAC) of cell chulture.
13. methods according to claim 12, for lysine monomethylation in identification of cell modifies substrate, cell can be cultivated and be added with in the amino acid whose SILAC nutrient culture media of stable isotope, and described isotope amino acid comprises 12c-arginine or 13c-arginine, 12c-lysine or 13c-lysine, 12cH 3-methionine or 13cD 3-methionine.
14. methods according to claim 13, cell chulture is containing 12cH 3-methionine or 12cD 3in the SILAC nutrient culture media of-methionine, both can carry out the modification of lysine monomethylation Qualitative Identification ( 13cD 3-mark), also can carry out the modification of lysine monomethylation quantitative test ( 12cH 3-or 13cD 3-mark).
15. according to the method one of claim 1-14 Suo Shu, and the protein being extracted from any cell, tissue or body fluid can carry out external chemical derivatization reaction; Preferentially, external chemical derivatization reaction is the acylation reaction on protein lysine residues ε-amido; More preferentially, the acylation reaction on protein lysine residues ε-amido is propionating derivative reaction.
16. methods according to claim 5-8 or 15, the albumen of substrate protein or external propionyl derivatization carries out protease digestion and produces peptide hydrolysis; Preferentially, proteinase is trypsase (trypsin).
17. methods described by claim 16, the enzymolysis polypeptide of generation can be separated into multiple polypeptide fractions by high performance liquid chromatography (HPLC) further; Preferentially, alkaline high performance liquid chromatography (basicHPLC) is adopted to be separated.
18. methods according to claim 1, propionyl methylates, and the chemical crosslinking of lysine ubiquitin antibody, covalent bond or Non-covalent binding are any not to be destroyed in the matrix of Antibody avidity, wherein, preferentially, Non-covalent binding is on the agarose resin of albumin A or Protein G or albumin A/G; More preferentially, Non-covalent binding is prepared into antibody coupling resin on Protein-A Sepharose glucoresin.
19. according to claim 1 and method according to claim 18, and other acyls methylate, and the chemical crosslinking of lysine ubiquitin antibody, covalent bond or Non-covalent binding are any not to be destroyed in the matrix of Antibody avidity.Preferentially, Non-covalent binding is on the agarose resin of albumin A or Protein G or albumin A/G; More preferentially, Non-covalent binding is prepared into antibody coupling resin on Protein-A Sepharose glucoresin.
20. methods according to claim 1, whether the lysine ε-amido being judged antibody affine enrichment peptide section by the result of Mass Spectrometric Identification and MASS SPECTRAL DATA ANALYSIS exists propionyl to methylate modification, comprise the albumen that decorating site, the sequence of modified polypeptide and modified polypeptide are corresponding.
21. profits require the method described in 20, mass spectrophotometry can comprise Matrix Assisted Laser Desorption time-of-flight mass spectrometry and analyze (MALDI-TOF), receives and rises liquid phase Tandem Mass Spectrometry Analysis (Nano-LC-MS/MS) or three grades of mass spectrums (MS/MS/MS) analyses.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105693816A (en) * 2016-03-17 2016-06-22 复旦大学 Di/trimethylation peptide enrichment and mass spectrum analysis method
CN105837677A (en) * 2016-04-15 2016-08-10 上海交通大学 Modified protein, and preparation method and application thereof
CN108072555A (en) * 2016-11-16 2018-05-25 中国科学院大连化学物理研究所 A kind of analysis and processing method of protein methylation and its application
CN108445221A (en) * 2017-02-16 2018-08-24 中国科学院大连化学物理研究所 A kind of identification method of protein methylation
CN110824168A (en) * 2018-08-10 2020-02-21 天津医科大学 Polypeptide probe and application thereof in identification of binding protein modified after translation
CN112852914A (en) * 2019-11-28 2021-05-28 中国科学院大连化学物理研究所 Method for weakening drug resistance of tumor cells
CN112961236A (en) * 2021-02-08 2021-06-15 深圳大学 Methylation modified KRAS4B protein and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005049636A2 (en) * 2003-11-21 2005-06-02 Nec Corp Method of analyzing protein
CN102375065A (en) * 2010-05-27 2012-03-14 杭州景杰生物科技有限公司 Identifying Method For Acylation Of Lysine Butane In Protein And Preparation Of Affine Reagent Thereof
CN103183725A (en) * 2011-12-27 2013-07-03 杭州景杰生物科技有限公司 A method of detecting protein lysine crotonylation acylation modification and developing affinity reagents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005049636A2 (en) * 2003-11-21 2005-06-02 Nec Corp Method of analyzing protein
CN102375065A (en) * 2010-05-27 2012-03-14 杭州景杰生物科技有限公司 Identifying Method For Acylation Of Lysine Butane In Protein And Preparation Of Affine Reagent Thereof
CN103183725A (en) * 2011-12-27 2013-07-03 杭州景杰生物科技有限公司 A method of detecting protein lysine crotonylation acylation modification and developing affinity reagents

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BENJAMIN A GARCIA, ET AL.: "Chemical derivatization of histones for facilitated analysis by mass spectrometry", 《NATURE PROTOCOLS》 *
SHAO-EN ONG, ET AL.: "Identifying and quantifying in vivo methylation sites by heavy methyl SILAC", 《NATURE METHODS》 *

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Publication number Priority date Publication date Assignee Title
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CN105837677B (en) * 2016-04-15 2020-12-25 上海交通大学 Modified protein and preparation method and application thereof
CN108072555A (en) * 2016-11-16 2018-05-25 中国科学院大连化学物理研究所 A kind of analysis and processing method of protein methylation and its application
CN108445221A (en) * 2017-02-16 2018-08-24 中国科学院大连化学物理研究所 A kind of identification method of protein methylation
CN108445221B (en) * 2017-02-16 2020-06-02 中国科学院大连化学物理研究所 Identification method of protein methylation
CN110824168A (en) * 2018-08-10 2020-02-21 天津医科大学 Polypeptide probe and application thereof in identification of binding protein modified after translation
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