CN105277712B

CN105277712B - A kind of method for identifying the modification of lysine ε amino side chains monomethylation

Info

Publication number: CN105277712B
Application number: CN201410360062.3A
Authority: CN
Inventors: 程仲毅
Original assignee: PTM Biolabs Inc
Current assignee: Hangzhou Jingjie Biotechnology Co.,Ltd.
Priority date: 2014-07-25
Filing date: 2014-07-25
Publication date: 2017-07-11
Anticipated expiration: 2034-07-25
Also published as: CN105277712A

Abstract

The present invention provides a kind of method for identifying lysine monomethylation modification substrate, and method is comprised the following steps：1) using external acylated derivative reaction, substrate protein lysine residue monomethylation ε amidos are derived as acyl methylates ε amidos；2) acyl prepared using the specificity affine enrichment propionyl of lysine ubiquitin antibody that methylates is methylated the peptide fragment of modification；3) acyl of the affine enrichment of Mass Spectrometric Identification antibody methylates site, polypeptide sequence and the substrate protein of modification.

Description

A kind of method for identifying the modification of Lysine s-amino groups side chain monomethylation

Technical field

The invention belongs to checkout and diagnosis field, especially, belong to lysine list in a kind of identification and quantitative cell or tissue Methylate the method for modifying substrate；More particularly, the protein groups using affine enrichment Yu the mass spectral analysis of specific antibody are belonged to Lysine monomethylation modification substrate in cell or tissue is identified and quantified to method.

Background technology

The epsilon-amino side chain of protein lysine residues can occur single, double or tri-methylated modification.Past tens Nian Li, the biological study of the modification (Kme) that methylated to lysine is concentrated mainly on core histones.The research of early stage is demonstrated Key effect of this modification in chromosome structure and function are exercised.This decorating state has opposite catalysis by 2 groups Enzyme adjusted, i.e. lysine methylated transferase and lysine demethylase.At present 50 lysines are had more than to methylate Transferase and about 25 lysine demethylases are found.Histone come propylhomoserin methylate modification it is related to various diseases, for example Cancer.Accordingly, lysine methylates regulation enzyme as a series of potential drug targets.

The current protein post-translational modification type (PTMs) found in histone all exists in nonhistones. The lysine for identifying methylates in regulation enzyme, there is the enzyme of some acellular nuclear locations and newfound not with histone as bottom The lysine of thing methylates regulation enzyme, and illustrating that lysine methylates should be widely distributed in nonhistones.Identification protein bottom Thing is to determine the premise of protein post-translational modification function, lysine methylate biology research history by the important of the premise Property illustrate be perfectly clear.Found after integrating we and other people data, lysine acetylation substrate appear in chromosome structure, In transcriptional regulatory and such as metabolic three research fields, and the plyability with substrate each other.Although lysine second The Substrate Identification of acylated modification is slower, and the research on it shows that lysine acetylation is collaboration in above three research field Effect.Similarly, lysine methylates the identification of modification group and quantitative comparison is found that the unrelated with chromosome of some downstreams Nonhistones and signal path, this lays a solid foundation for follow-up functional study.

Even so, identification lysine methylated substrate and remarkable.Introduced with identification phosphorylation modification³²P is marked not Together,³H or¹⁴The low-activity of C makes radio isotope detection method be difficult to carry out in the identification of lysine methylated substrate.First Base, especially monomethylation (Kme1), only introduce a building stone for very little in its substrate, thus relying of being modified Propylhomoserin group is trickle with the sine group difference modified, and the minimum conformation of only one of which resists for developing compatibility Body.To take into account compatibility and specificity simultaneously, develop the methylate antibody (ubiquitin antibody) unrelated with sequence and be one and huge choose War.As a result, because the peptide fragment enrichment method that do not methylate suitably is for follow-up mass spectral analysis provides peptide fragment, lysine first The identification process of base substrate is slow.As described in phosphorylation and the research of lysine acetylation group, affine enrichment is research Committed step (Kim, S.C.et the al.Substrate and of protein post-translational modification (PTM) global analysis functional diversity of lysine acetylation revealed by a proteomics survey.Mol Cell23,607-618(2006)).Due to the lysine and the thing of unmodified lysine of monomethylation modification Change nature difference very little, therefore using such as isolation of phosphorylated polypeptide immobilized metal affinity chromatography (IMAC) chemical method come Separate highly difficult (Ficarro, S.B.et the al.Phosphoproteome analysis of lysine polypeptide of the modification that methylates by mass spectrometry and its application to Saccharomyces cerevisiae.Nature biotechnology20,301-305(2002)).In addition, preparing the anti-lysine monomethylation of high specific and high-affinity Antibody it is also highly difficult.Accordingly, it would be desirable to carry out innovative ability based on proteomic techniques lysine is methylated to existing Modification is identified and analyzed.Especially, it is necessary to enter with quantitative analysis method to the Substrate Identification of lysine monomethylation modification The brand-new innovation and creation of row.

The content of the invention

To solve this technical difficulty, the present invention develops a new chemical proteomics method.Using the method Can efficiently, accurately identify in peptide fragment whether the site of lysine ε-amido monomethylation modification and modification, it is quantitative Analyze the change level of modification.

On the one hand, whether the present invention there is list in providing a kind of identification substrate peptide fragment or albumen on the ε-amido of lysine Methylate the method for modification, and method includes：Using external nitrogen acylation derivative reaction, the monomethylation ε allowed on described substrate- The lysine residue of amido derives as acyl methylates ε-amido；There is acyl and methylate in the affine enrichment of ubiquitin antibody methylated with anti-acyl The peptide fragment of modification；Identify whether the polypeptide of the affine enrichment of antibody there occurs that propionyl methylates modification with LC-MS mass spectrograph.

Preferably, the result for being obtained according to the detection of LC-MS mass spectrograph, and thus judge or estimate substrate protein or peptide Whether the lysine list firstization of physical presence is modified in section.

Preferably, the carbon of the modification group of nitrogen acylation modification is carried out to substrate protein lysine residue monomethylation ε-amido Total atom number is less than 8.Preferred the total number of carbon atoms is less than 6.Preferably, the modification group can include alkyl or fragrance The total number of carbon atoms of base, wherein alkyl or aromatic radical is less than 8.Preferably, the total number of carbon atoms of alkyl is less than 6, or, fragrant The total number of carbon atoms of perfume base is less than 8.

Preferably, the lysine residue of amido is allowed to derive as acetonyl ε-amido, butyryl methylates ε-amido, isobutyl Acyl methylates ε-amido, and valeryl methylates ε-amido, and 2- methylbutyryls methylate ε-amido, 3- methylbutyryls methylate ε- Amido, 2,2- dimethyl propylenes are acylated ε-amido, succinylation methylates ε-amido or malonyl methylates ε-amido etc..Acyl Changing reagent includes various carboxylic acids, acid anhydrides, acyl chlorides, and other all acylating reagents.

Preferably, acylating reagent is the acylating reagent with stable isotope.Isotope acylating reagent be carbon containing, hydrogen, oxygen, The carboxylic acid of nitrogen stable isotope, acid anhydrides, acyl chlorides, carboxylate, acid amides and other all acylating reagents.Preferentially, same position is stablized Plain reagent is¹²C₆H₁₀O₃Propionic andydride,¹³C₆H₁₀O₃Propionic andydride or¹²C₆D₁₀O₃Propionic andydride.

In other preferred modes, allow substrate protein or peptide to carry out nitrogen acylation reaction is carried out in vitro.It is excellent at some In the mode of choosing, the acylation reaction of substrate protein or peptide can be carried out before or after the protease digestion substrate.Preferably, Substrate protein or polypeptide are extracted from any cell, tissue or body fluid.Described substrate protein or polypeptide are total protein or total many Peptide.

In some preferred modes, total protein is extracted from cell or tissue with propionyl acid anhydride in the carbonic acid that pH value is 8.5 There is chemical reaction in ammonium-sodium bicarbonate buffer liquid and produce the propionating external derived protein of lysine, through pancreas egg after reaction completely White enzymic digestion produces the propionating derivative enzymolysis polypeptide of lysine.In some preferred modes, propionating reaction is prepared complete The method of the propionating external derived protein of lysine includes：200 μ L propionic andydrides are quickly adding into the cell containing 20mg total proteins In lysate, shake and will add appropriate 2M NaOH that pH is adjusted into 8, then in incubation at room temperature 30 minutes；Reacted lysine Propionating external derived protein is precipitated by trichloroacetic acid (TCA) precipitation method.Further to produce the propionating derivative enzyme of lysine Solution polypeptide by protein precipitation 2ml digestion buffer solution (0.1M NH₄HCO₃, pH=8.5) in it is resuspended, it is then slow with digestion is dissolved in Fliud flushing trypsase (trypsin) digestible protein 16 hours；After digestion completely, it was centrifuged off by 10 minutes of 20,000x g Precipitation, obtains the propionating derivative enzymolysis polypeptide supernatant of lysine.In preferred mode, pancreatin and the propionating external derivative of lysine The ratio of albumen is 1:100(w/w).

In vitro in lysine derivative reaction, the ε-amido of unmodified lysine or monomethylation modification lysine There is propionating reaction, generate propionating lysine or propionyl methylates lysine, and di-methylation and tri-methylated bad ammonia Acid can not but occur above-mentioned reaction.If propionating reaction is complete in theory, will not be deposited on the lysine residue of albumen after reaction In unmodified lysine.Therefore, after reacted albumen being produced into enzymolysis polypeptide through enzymolysis, identified by mass spectral analysis and rely ammonia There is no the polypeptide of any modification on sour residue, compare the type polypeptide in all lysine residue modified polypeptides for identifying Ratio can assess the reaction efficiency of propionating reaction..

Some preferred embodiment in, described albumen (total protein or total polypeptide, or specific albumen or many Peptide) before propionating reaction is carried out, it is extracted from the tumour cell or clinical sample by cell culture medium culture.Preferably, Cell is the tumour cell of the immortality passage that can carry out the cold labeling (SILAC) based on cell culture.It is furthermore preferred that It is lysine monomethylation modification substrate in identification of cell, cell can be cultivated and be added with the SILAC of stable isotope amino acid In culture medium.Preferentially, isotope amino acid includes¹²C- arginine or¹³C- arginine, more preferentially,¹²C- lysines or¹³C- lysines, more preferentially, stable isotope amino acid is¹²CH₃- methionine or¹³CD₃- methionine.It is preferred at some Mode in, be in vitro provide lysine methylate modification methyl source, be with the addition of in cell culture medium¹²CH₃- first sulphur ammonia Acid (¹²CH₃-Met)；Or, add in the medium¹³CD₃- methionine (¹³CD₃-Met).Herein, methyl on methionine In group¹²C is by stable isotope¹³C is replaced, H₃(hydrogen) is by the D of stable isotope₃(deuterium) is replaced.

Methylated in a pair of cells of quantitative comparison modification change when, it is necessary to a cell culture is being contained¹²CH₃- first In the culture medium of methyllanthionine, another cell culture is in cold labeling¹³CD₃In the culture medium of-methionine, the party Method is referred to as amino acid cold labeling strategy (SILAC) (the Ong SE and Mann M.A based on cell culture practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat Protoc.1(6):2650-60.(2006))。

Some preferred embodiment in, described cell derived in commercially available cell line, selected from K562 cells (chronic myelogenous leukemia cell line), SW620 cells (colon carcinoma cell line), A549 cells (Lines) and One or more in SMM7721 cells (HCC).It is tissue-derived for clinical diagnosis for liver cancer patient surgery excision liver Cancerous tissue sample.

Some preferred embodiment in, it is necessary to carry out specific anti-nitrogen acyl to there is the methylate peptide fragment of modification of nitrogen acyl Methylate the affine enrichment of ubiquitin antibody.In preferred mode, nitrogen acyl methylates lysine ubiquitin antibody in advance by being chemically crosslinked, being total to In valency combination or any matrix for not destroying antibody compatibility of Non-covalent binding.Described matrix is agarose resin, by non- It covalently bind on the agarose resin of albumin A or Protein G or albumin A/G；More preferentially, Non-covalent binding is in Protein-A Sepharose Antibody coupling resin is prepared on glucoresin.

Preferably, described ubiquitin antibody is affine to antibody is not destroyed by chemical crosslinking, covalent bond or Non-covalent binding In the matrix of property.Preferentially, by Non-covalent binding on the agarose resin of albumin A or Protein G or albumin A/G.More preferably Ground, Non-covalent binding is prepared into antibody coupling resin on Protein-A Sepharose glucoresin.

In some preferred modes, the ubiquitin antibody that described antibody methylates for specific bond propionyl.The enrichment method Comprise the following steps：The general propionyl agarose resin that ubiquitin antibody is crosslinked with albumin A that methylates is coupled, albumin A is prepared and is resisted Body binding resin.In some embodiments, affine enrichment methylates propionyl ubiquitin antibody and bad ammonia under the conditions of being additionally included in 4 DEG C The propionating derivative polypeptide night incubation of acid.Or using antibody coupling resin and the propionating derivative polypeptide night incubation of lysine, Then 1ml NETN buffer solutions (100mM NaCl, 1mM EDTA, 20mM Tris pH8.0and0.5% (w/v) NP-40) are used Antibody coupling resin after washing incubation；Then the methylate peptide fragment of modification of the propionyl of affine enrichment in antibody resin is used 100 0.1% (v/v) trifluoroacetic acid (TFA) of μ l is washed by 3 times；Eluent merges and then is drained with concentrating instrument.

Some preferred embodiment in, before the peptide fragment that is modified is identified with LC-MS mass spectrograph, to carrying out third The peptide hydrolysis of albumen carry out HPLC separation after acylated derivative reaction, the propionating derivative polypeptide fractions of the lysine after separation point Propionyl is not carried out to methylate the affine enrichment of ubiquitin antibody.Preferably, ionization treatment is carried out to the polypeptide being enriched with, is then carried out The mass spectral analysis of Nano-HPLC/MS/MS.Analyzed through specific MASS SPECTRAL DATA ANALYSIS software (Mascot or Maxquant), identification Go out specific propionyl on lysine residue to methylate the corresponding mass shift of modification, and thereby determine that on polypeptide lysine residue Monomethylation decorating site, the quantitative change of modification, modified polypeptide sequence and the corresponding albumen of the polypeptide sequence.It is right finally to obtain Lysine monomethylation repaiies the result of the identification of substrate and the quantitative change of feature site modification level.

Beneficial effect

By this method, can accurately, specifically in substrate protein lysine monomethylation modification be identified and Carry out the quantitative analysis of lysine monomethylation modification., by the method for the present invention, we authenticate in 403 albumen for this 460 monomethylation sites, with accuracy very high, have obtained lysine monomethylation spectrum maximum so far.This 460 There is fraction to be reported by former research institute in individual monomethylation site, it was confirmed that the validity of the inventive method.And it is most of It is the site of lysine monomethylation modification unknown in the past, lysine monomethylation modification is recognized so as to has greatly enriched Know.Lysine of the present invention methylates the function in intracellular approach, metabolism network and composite construction of the analysis shows modifications of group. Our result provides abundant data resource for the functional study of follow-up lysine monomethylation path.

Brief description of the drawings

Fig. 1 is the experimental design process figure of identification lysine monomethylation polypeptide in one specific implementation example of the present invention. Figure 1A is that (the propionating external derivative reaction of lysine, antibody is affine for experiment flow schematic diagram of the identification containing monomethylation polypeptide Enrichment and mass spectral analysis)；Figure 1B is conventional acylating reagent type；Fig. 1 C are with the propionating external derivative reaction of lysine, resist Experiment flow schematic diagram of the identification containing monomethylation polypeptide as a example by enrichment that body is affine and mass spectral analysis；Fig. 1 D are specific to produce The chemical synthesising technology route map of the propionyl monomethylation lysine of propionyl monomethylation lysine ubiquitin antibody；Fig. 1 E print for spot Mark (dot-spot) detects the specificity experiments of 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 generation Table lysine crotons are acylated peptide library.Fig. 1 F are a lysine propionyl methylated polypeptides from HSP90 albumenTypical mass spectral analysis annotated map.pr:Propionylation, it is propionating；me: Methylation, methylates；pr+me:Propionyl methylation, propionyl methylates；bu:Butyrylation, fourth It is acylated；me2:Di-methylation, di-methylation；me3:Tri-methylation, it is tri-methylated；cr: Crotonylation, crotons are acylated

The external propionyl derivative reaction Efficiency testing of Fig. 2 lysines.K562 cell holoprotein lysates are external through lysine After propionyl derivative reaction, pancreatin digestion produces enzymolysis polypeptide, the propionating lysine in mass spectral analysis detection enzymolysis polypeptide.Rely The propionating efficiency of propylhomoserin by propionating lysine polypeptide, estimate by the ratio in all polypeptides for identifying.

Fig. 3 lysine propionyl methylate decorative features spectrum.Fig. 3 A be through¹³CD₃The typical propionyl of-Met marks methylates and repaiies The MS spectrograms of the polypeptide of decorations.Fig. 3 B are the YSQADALK of EZH2 albumen in HeLa cells_pro-meThe MS/MS mass spectrums of YVGIER polypeptides Figure.Top be by¹²CH₃The YSQADALK of-mark_pr+meYVGIER peptide fragment MS/MS mass spectrograms, wherein, it is below¹³CD₃- markThe mass spectrogram of polypeptide, shows the b- of same propionyl methylated polypeptides, and y- ions exist 4Da mass transfers after cold labeling.Fig. 3 C be from " light " (¹²CH₃-) " weight " (¹³CD₃-) methionine mark The methylated polypeptides number comparison diagram identified in HeLa cell pyrolysis liquid mixtures.Alkaline HPLC column of Fig. 3 D displays is separated and obtained 20 polypeptide fractions methylated through general propionyl Mass Spectrometric Identification is arrived after antibody richness the quantity of lysine monomethylation polypeptide and The bioaccumulation efficiency of antibody.pr:Propionylation, it is propionating；me:Methylation, methylates；pr+me:propionyl Methylation, propionyl methylates.

The protein lysine monomethylation group of Fig. 4 high confidence levels.Fig. 4 a are all 463 identified in all samples The Mascot ion score distribution maps of monomethylation polypeptide；Only show polypeptide of the Mascot ions score higher than 30.Fig. 4 b are from 5 The lysine monomethylation polypeptide number identified in individual tumor cell line Wien comparison diagram (HeLa (cervical cancer cell), K562 (chronic myeloid leukemia cells), SW620 (colon cancer cell), (liver cancer is thin for A549 (lung carcinoma cell) and SMM7721 Born of the same parents).

In Fig. 5 K562 cells on CDC5L albumen K114 propionyl methylation sites mass spectrum proof diagram.Fig. 5 a are synthesis Propionyl methylated polypeptides LANTQGK_pr+meK_prAK_prR Tandem Mass Spectrometry Analysis, with the phase homopolypeptide table arrived from K562 cellular identifications Reveal identical ionic strength figure, the top of figure is polypeptide LANTQGK in vivo by present invention identification_pr+meK_prAK_prThe matter of R Spectrogram, lower section is the mass spectrogram of the polypeptide of synthesis.Fig. 5 b are internal polypeptide LANTQGK_meK and its external synthesis polypeptide LANTQGK_meThe Tandem Mass Spectrometry Analysis of K.Y-ion and b-ion parts 4Da is migrated, and the position with lysine methyl residues migrates one Cause.pr:Propionylation, it is propionating；me:Methylation, methylates；pr+me:propionyl Methylation, propionyl methylates.

The mass spectrum checking of Fig. 6 lysine propionyl methylated polypeptides.Mass spectrum annotated map comes from intracellular lysine propionyl first The contrast of base polypeptide and corresponding synthesis polypeptide.Wherein, Fig. 6 A are KHDRSB1 Identification of Fusion Protein PVK in HeLa cells_pr+meGAYR (the wherein K of peptide fragment_pr+meRepresent the lysine of propionyl monomethylation) second order mses (MS/MS) figure.Fig. 6 B are corresponding external conjunction Into the mass spectrogram (PVK of polypeptide_pr+meGAYR).Fig. 6 C are the K of K562 cell EIF4H Identification of Fusion Protein_pr+me(its of GGPDDR peptide fragments Middle K_pr+meRepresent the lysine of propionyl monomethylation) second order mses (MS/MS)；Fig. 6 D are two grades of corresponding external synthesis polypeptide Mass spectrogram (K_pr+meGGPDDR).pr:Propionylation, it is propionating；me:Methylation, methylates；pr+me: Propionyl methylation, propionyl methylates.

Fig. 7 is the decorating site figure that the present invention is identified different cells, and ((cervical carcinoma is thin for HeLa for 5 tumor cell lines Born of the same parents), K562 (chronic myeloid leukemia cells), SW620 (colon cancer cell), A549 (lung carcinoma cell)) and SMM7721 (livers Cancer cell) in 29 lysine monomethylation modified protein lists all identifying.

Describe in detail

The substrate mark of lysine monomethylation modification

The modification that methylates of amino acid is a kind of abiogenous phenomenon of life entity or process.Based on current research table Bright, in cell body, S- adenosines-L-Met (SAM) is the most important direct donor of methyl group, and first thiamine (Methionine, Met) is the direct premise for synthesizing SAM.Therefore, when methionine is added in the medium, by cell After internal series reaction approach, the methyl group (CH on methionine₃-) formation on the ε-amido of lysine will be transferred to Methylate modification.Which constitute lysine methylate modification substrate mark General Principle, i.e., added not in cell culture medium With the first thiamine of cold labeling, such as¹²CH₃- Met or¹³CD₃- Met, then will make on substrate protein band respectively¹²CH₃- Methyl group or¹³CD₃The methyl group of-Met.Mark approach is summarized as follows：

A:¹²CH₃- methionine → S- adenosine-L methionine-^-12CH₃(SAM) → lysine-¹²CH₃(lysine monomethyl Change)

B:¹³CD₃- methionine → S- adenosine-L methionine-^-13CD₃(SAM) → lysine-¹³CD₃(lysine monomethyl Change).

For containing a same sequences polypeptide for lysine monomethylation modification, use¹²CH₃- Met or¹³CD₃- Met mark only difference is that, due to the difference of the molecular weight of stable isotope,¹³CD₃The polypeptide of-Met marks will compare¹²CH₃Same sequences polypeptide weight 4.02Da (Fig. 3 a) of-Met marks, so that being capable of changing by mass shift in mass spectral analysis Change this two polypeptides are distinguished, and polypeptide amount in itself number can be reflected by the abundance of mass spectral analysis.This The theoretical foundation of the same albumen of analysis protein modification quantitative analysis at different conditions is constituted, referred to as based on cell culture Amino acid cold labeling strategy (SILAC) (Ong SE and Mann M.A practical recipe for stable isotope labeling by amino acids in cell culture(SILAC).Nat Protoc.1 (6):2650-60.(2006)).For the qualitative analysis of the lysine monomethylation modification substrate in cell or tissue, nothing The labelling strategies of stable isotope must be introduced, is directly to be capable of achieving by the method for antibody enrichment, mass spectral analysis.And for quantitative When analyzing lysine monomethylation modification level in a pair of consistent cell samples of genetic background, then need to introduce¹²CH₃-Met Or¹³CD₃The SILAC strategies of-Met marks, are then realized by the method for antibody enrichment, mass spectral analysis.

For how to methylate and repair come certain protein modification of qualitative or quantitative analysis, including lysine using mass-spectrometric technique Decorations, the technical method that persons skilled in the art are known.

The propionating derivative reaction of lysine monomethylation modification

Under normal circumstances, the identification of certain protein modification substrate firstly the need of with the specific ubiquitin antibody of the modification to tool Having the modified polypeptide carries out affine enrichment, such as identification of acetylation substrate.But for the modification of lysine monomethylation, by In the too small (CH of methyl group₃-) and very weak immunogenicity, it is difficult to directly obtain the general anti-of lysine monomethylation modification Body.In order to overcome the difficulty, inventor envisions can occur lysine ε-amido that lysine monomethylation is modified with specificity It is upper that extra group is introduced by external derivative reaction, so as to increase the space structure of lysine ε-amido modification group, be The preparation of antibody provides bigger determinant and stronger immunogenicity.

The propionating reaction of lysine is the external derivatization that a kind of chemical reaction and synthesis skilled person know Reaction, by being reacted under propionic andydride and substrate protein in vitro special reaction condition, can be in the lysine of substrate protein ε-amido On be artificially introduced propionyl group, form the propionating lysine of derivatization.For the modification of lysine monomethylation, due to relying Still there is an extra propionyl anhydride reactant site on propylhomoserin ε-amido, therefore propionating reaction can also occur, produce derivatization Propionyl methylate lysine (Fig. 1 a).And ε-the amido on di-methylation modification or tri-methylated modification lysine is not available for Propionyl anhydride reactant site can not produce propionyl derivative reaction (Garcia, B.A.et al.Chemical derivatization of histones for facilitated analysis by mass spectrometry.Nature protocols2, 933-938 (2007)) methylated when the modification of internal lysine monomethylation forms lysine propionyl through external derivative reaction During modification, modification group is greatly increased on space structure, it is easier to by antibody institute specific recognition.Therefore can make in theory A standby species specificity is known propionyl and is methylated the antibody of lysine, for recognizing by the lysine monomethyl of propionating derivatization reaction Change modified polypeptide (Fig. 1 c).

Garcia and the people of Hunter two first by lysine it is propionating be applied to histone (first pass through extraction histone, Then to extraction after 5 kinds of histones to carry out lysine propionating) (Garcia, B.A.et al.Chemical in modification research derivatization of histones for facilitated analysis by mass Spectrometry.Nature protocols2,933-938 (2007)) this chemical method was not tested at that time more multiple In miscellaneous system, whether the total protein such as in cell or tissue lysate is feasible.However, the present invention is to the total of cell or tissue Albumen is extracted first, then carries out propionating reaction in vitro again.After through protease cracking, using antibody enrichment and mass spectrum point The method of analysis is analyzed (detailed description sees below) in holoprotein group aspect to lysine monomethylation group.

In a preferred mode, the K562 cell pyrolysis liquids holoprotein that the present invention is used is in pH value with propionic andydride Chemically reacted under conditions of 8.5.After reaction, after being digested in pH7.2 ammonium carbonates-sodium bicarbonate buffer liquid with pancreatin, through mass spectrum There is the polypeptide of the propionating reaction of lysine in analysis.If external propionating reaction is complete, the ratio containing unmodified lysine polypeptide Example will be very low.Qualification result shows, only has 37 polypeptides (to account for after propionating reaction, in all 2,556 polypeptides for detecting 1.4%) contain unmodified lysine in, illustrate the external propionating reaction used in the present invention for complicated shell egg lean type There is efficiency (Fig. 2) very high in system.

Either use¹²CH₃- Met or¹³CD₃- Met is marked to cell, mark¹²CH₃- or¹³CD₃- lysine list The modification that methylates can occur propionating derivative reaction, generate propionyl¹²CH₃- lysine or propionyl¹³CD₃- lysine.For For introducing propionating derivative reaction come the modification of qualitative or quantitative analysis lysine monomethylation, use¹³CD₃- Met is marked Another necessary reason of note is, due to propionyl¹²CH₃Mass shift produced by-lysine is as lysine Butyrylation (70.0440Da), and propionyl¹³CD₃- lysine but the mass shift with unique 74.0640Da, so as in mass spectral analysis Internal lysine Butyrylation modification can be distinguished, the effect of precise Identification lysine monomethylation modification is reached.

The enrichment of lysine propionyl methylated polypeptides and the preparation of ubiquitin antibody

Described in phosphorylation and the research of lysine acetylation group, affine enrichment is that research protein modification integrally divides The committed step of analysis¹¹.The physico-chemical property difference very little of lysine and unmodified lysine is modified due to monomethylation, therefore is used Such as immobilized metal affinity chromatography (IMAC) chemical method of isolation of phosphorylated polypeptide separates the lysine of the modification that methylates Polypeptide is highly difficult¹⁵.In addition, the antibody for preparing the lysine monomethylation of high specific and certain compatibility is also highly difficult.It is solution Certainly this is difficult, and the method that the present invention develops a kind of enrichment monomethylation polypeptide of novelty, the method includes following three master Want step：(1) by the ε amino of monomethylation on lysine and propionic acid anhydride reactant, the propionyl ε amino that methylates is formed chemically derived Thing；(2) methylated containing propionyl the polypeptide of lysine using the lysine antibody enrichment that methylates of specific propionyl；(3) efficient liquid phase The polypeptide being enriched with mass spectral analysis, the sequence and determination decorating site of identification antibody enrichment polypeptide.

The external propionating derivatization reaction of lysine that introduces is the premise based on antibody enrichment method, and its principle is with importance In preceding detailed description.It is equally important that the inventive method needs to use specific propionyl methylating the ubiquitin antibody of lysine.Due to third Acyl methylated groups prepare the methylate ubiquitin antibody of lysine of propionyl and compare that to prepare monomethylation lysine general anti-than big Body is simple.To confirm this it is assumed that immune rabbit after being coupled with propionyl monomethylation lysine (Fig. 1 D) and KLH, then from Anti- propionyl is purified into rabbit anteserum to methylate lysine ubiquitin antibody.Contain propionating lysine (K using fixed position_pr), Butyrylation Lysine (K_buty), crotons acylated lysine (K_cr), monomethylation lysine (Kme1), di-methylation lysine (Kme2) and three Methylate lysine (Kme3) peptide library Dot blot (dot blot) method determine antibody specificity (Fig. 1 c).As a result table It is bright, the propionating lysine ubiquitin antibody that methylates propionyl is methylated lysine peptide library specificity it is stronger than other peptide libraries by 100 More than times.Although propionyl methylates, the structure of lysine is similar with the structure of propionating lysine and Butyrylation lysine, spot The propionyl that the testing result of point trace shows used in the present invention methylate lysine ubiquitin antibody with compatibility very high and Specificity, can be used for the enrichment of lysine propionyl methylated polypeptides.

Affine enrichment based on antibody to modified polypeptide is one of main method of high throughput protein modification group, such as Had a wide range of applications in lysine acetylation research.When the total protein extracted from cell or tissue is through external propionating anti- Ying Hou, can produce the propionating derivative polypeptide of lysine, including lysine propionyl to methylate derivative polypeptide through protease digestion. In one preferred mode, protease is mainly trypsase (trypsin).Then, specific propionyl is methylated lysine Ubiquitin antibody prepares antibody coupling resin with albumin A (Protein A) agarose resin covalent coupling, by antibody coupling resin in spy Determine derivative polypeptide propionating with lysine in buffer solution to be incubated, then the more specific affine combination characteristic of Ag-Ab, rely ammonia Sour propionyl methylates by derivative polypeptide affine combination in antibody resin.Lavation buffer solution washes away non-specific binding polypeptide Afterwards, the lysine propionyl of specific bond on the antibody derivative polypeptide that methylates can be eluted with the elution buffer of low ph value. Carried out after draining Nano-HPLC-MS/MS mass spectral analyses can identify lysine propionyl methylate derivative polypeptide sequence and determination repair Decorations site.

Propionyl monomethylation lysine polypeptide is detected

Chemical modification group can make the mass shift of modification substrate and polypeptide, such as-CH of monomethyl modification₂Group can make bottom There is the mass shift of 14.0147Da in thing, and phosphorylation modification can make substrate that the mass shift of 79.9663Da occurs.Lysine As the mass shift that occurs with some amino acid mutations of mass shift of generation that methylates, such as serine is for being mutated into Soviet Union's ammonia Acid, for glutamic acid is mutated into, for leucine or isoleucine is mutated into, asparagine is replaced and is mutated into paddy ammonia valine aspartic acid Acid amides.Make lysine that identical mass shift to occur with Butyrylation in addition, propionyl methylate.Lysine Butyrylation is also internal egg A kind of modification mode of white matter.Thus, whether there is monomethylation modification on to certain Specific amino acid or base occurs in vitro When group's modification is detected, if without correct quality control, identification propionyl methylated polypeptides are just likely occurred false sun The result of property.Therefore, this law is bright using existing¹³CD₃The method of isotope marks methylated polypeptides can avoid the occurrence of false positive Result.

It is for example when whether there is monomethylation modification on lysine is identified, thin using isotope labelling method culture During born of the same parents, by the methionine in conventional medium¹²CH₃It is substituted for¹³CD₃.In cell, it is known that lysine methylates precursor-S- glands Glycosides methionine (SAM) converts to be formed from methionine.Therefore, lysine methylates in cell,¹³CD₃Methionine turns Chemical conversion¹³CD₃S-adenosylmethionine (¹³CD₃- SAM), cause lysine that 18.027Da mass shifts occur.¹³CD₃- methylate There is propionating rear generation 74.0640Da mass shifts in vitro in lysine.And Mass Spectrometer Method can be inclined by this unique quality The mass shift occurred with other any of protein modification types or amino acid mutation is moved to distinguish.Such as in the present invention One embodiment in, propionyl methylated polypeptides (Fig. 1 c) is identified from HSP90 albumen, by the matter of 74.0640Da Amount skew, can be accurately positioned on the 3rd lysine of this polypeptide and propionyl occurs methylate modification, and thus actually infer the position Point there occurs that lysine monomethylation is modified.

Further to verify the reliability of the method, will¹³CD₃Methionine labeling method is combined with SILAC methods.Experiment When, the parallel culture of HeLa cells is existed¹²CH₃- methionine and¹³CD₃In methionine culture medium, rower is entered to Methyl groups Note.After mark is complete, same amount of two kinds of crack proteins are mixed, external propionating derivative reaction, then pancreatin digestion system Standby enzymolysis polypeptide.To reduce the complexity of sample and improving the flux of mass spectral analysis, with preparative high performance liquid chromatography (HPLC) Instrument isolated polypeptide, prepares 20 polypeptide fractions altogether.To each polypeptide fractions for preparing, being methylated with general propionyl, lysine is general to be resisted Body carries out the affine enrichment of propionyl methylated polypeptides, nano-HPLC/MS/MS analyses after enrichment polypeptide wash-out.

During mass spectral analysis, occur lysine propionyl methylate modification polypeptide because in methyl group hydrogen ion with¹²C carbon ions Respectively be replaced deuterium (D) ion with¹³C carbon ions, it will produce the mass shift (Fig. 3 A) of 4.002 dalton.To ensure point The accuracy of analysis, 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 that 183 confidence levels high are identified from HeLa cells methylates modified polypeptide (Fig. 3 C).20 enzymolysis In polypeptide fractions, bioaccumulation efficiency (Fig. 3 D) between 3% to 41%.By artificial nucleus to initial data, wherein, 180 propionyl The modified polypeptide that methylates has a pair of precursor ions, corresponding to 190 decorating sites that methylate in 162 kinds of albumen.Only two , containing only " weight (heavy) " ion, a polypeptide is containing only " light (light) " " ion for individual polypeptide.Explanation¹³CD₃Replace methyl group It is reliable that method is used for monomethylation site atlas analysis and rejects false positive results.Also demonstrate that this method for identifying list Methyl modified polypeptide is reliable.

The scope of protein lysine monomethylation group

Then apply¹³CD₃Methionine labeling method have detected K562 cells (chronic myelogenous leukemia cell), SW620 Cell (colon cancer cell), in 4 kinds of people source tumour cells of A549 cells (lung carcinoma cell) and SMM7721 cells (HCC) Lysine monomethylation is modified.Peptide identification is more less than 20 using strict 1% false positive rate and rejecting MASCOT score values Peptide, and manual confirmation each collection of illustrative plates accuracy.By after above-mentioned strict quality control, the present invention is altogether thin in four tumours 448 lysine monomethylation modified polypeptides of nonredundancy, 460 sites in 403 albumen of correspondence are identified in born of the same parents.This In a little polypeptides, 73% Mascot score values are higher than 30 (Fig. 4 A).By retrieving public Uniprot databases, what is identified is big Not yet report in part monomethyl site.The monomethyl lysine polypeptide and the complete list of albumen that the present invention is identified are shown in Fig. 7.

The lysine monomethylation modification of core histones is widely studied, thus can be used to as good positive right According to.Consistent with estimated, the present invention identifies 12 histone monomethyl decorating sites altogether.It is widely studied except identifying Outside K4, K9, K27, K36, K79 of histone H 3 and the several methylation sites of the K20 of histone H 4, rare research is also identified The K18 monomethylation decorating sites of histone H 3.There is these lysine monomethylations modification position in 5 kinds of cells due to known Point, the method that the present invention has identified this detection lysine monomethylation modification of these sites explanation is reliable and stable.Weight Want, using method of the present invention, inventor also identifies from many known monomethylation modified proteins New decorating site.Such as WIZ albumen, in addition to the lysine K976 sites reported are identified, K1321 and K1463 is also identified Two kinds of new lysine monomethylation sites.

In order to further verify the validity of the inventive method, inventor then analyzes the bad ammonia in the liver cancer tissue of people Sour monomethyl is modified to study the substrate spectrum of lysine monomethylation modification in clinical tissue.In 5 kinds of tumour cells above Identify lysine monomethylation modification data to compare, in as a result showing 29 monomethylation modified polypeptides in liver cancer tissue 32 monomethylation sites all contain in this 5 kinds of cells, including 20 new lysine methylation sites and 6 group eggs The lysine monomethyl site (Fig. 7) of white H3, further demonstrates that the reliability of the inventive method

The newly checking of this case monomethylation decorating site of the bad ammonia of identification

Because the site-specific antibodie of most nonhistones lysine monomethylation modifications can not be commercialized Obtain, so as to cannot be tested the site of Mass Spectrometric Identification using the biochemical method of Western blotting (western blotting) Card.Therefore the lysine that Mass Spectrometric Identification goes out is verified present invention employs corresponding lysine monomethylation modified polypeptide is synthesized The method of monomethylation modified polypeptide.The intracellular lysine monomethylation polypeptide that Mass Spectrometric Identification goes out is artificial synthesized with corresponding Modified polypeptide, under the conditions of identical mass spectral analysis, it should obtain identical second order mses figure (MS/MS), this is identical The standard of polypeptide checking.Therefore, being in the polypeptide between 20 to 60, arbitrarily to pick 9 Mass Spectrometric Identifications and go out from Mascot score values Propionyl monomethylation polypeptide sequence and artificial synthesized.With in K562 cells propionyl has been identified on CDC5L albumen K114 sites Methylate as a example by modifying, by artificial synthesized LANTQGK_pr+meK_prAK_prThe mass spectral analysis of polypeptide, show synthesis polypeptide with After enrichment that antibody is affine Mass Spectrometric Identification to polypeptide in addition to the difference of the 4Da caused by isotope marks have it is completely the same Second order mses figure (Fig. 5 A).Using in addition to being verified using the method for synthesis polypeptide, CDC5L albumen is also demonstrated in the cell In lysine monomethylation modification.Endogenous CDC5L albumen is purified into from the K562 cells of culture by immunoprecipitation Mass spectral analysis is carried out after coming.As it was previously stated, with¹³CD₃- mark methyl group, the CDC5L albumen that then mass spectral analysis is purified.From The monomethylation polypeptide LANTQGK of K164 is identified in the CDC5L albumen of intracellular purifying¹³CD_3-meK.Further to verify this Individual site, by analyzing the collection of illustrative plates of corresponding synthesis polypeptide, shows the collection of illustrative plates with intracellular polypeptide except because isotope marks cause 4Da difference outside, basically identical (Fig. 5 B).

Same verification method also demonstrates the PVK of the KHDRSB1 Identification of Fusion Protein in HeLa cells_pr+meThe two of GAYR peptide fragments Level mass spectrogram (Fig. 6 A) and artificial synthesized PVK_pr+meThe second order mses figure (Fig. 6 A) of GAYR peptide fragments is consistent；In K562 cells The K of EIF4H Identification of Fusion Protein_pr+meThe second order mses figure (Fig. 6 C) of GGPDDR peptide fragments and corresponding external synthesis polypeptide K_pr+ _meThe second order mses figure (Fig. 6 D) of GGPDDR is consistent.Thus the identification that method provided by the present invention is capable of high confidence level is demonstrated Lysine monomethylation is modified.

Specific embodiment

The present invention is using specific embodiment come to the peptide modified with the presence or absence of lysine monomethyl in cell or tissue It is this to enumerate how the simply exemplary illustration method of the present invention is realized or albumen is identified or quantified, and Any limitation can not be played to the present invention.Those of ordinary skill in the art can be in the feelings without prejudice to marrow of the invention Any improvement and change is done under condition to the present invention, but this change is contained in the scope of claim of the invention In.

Examples of implementation 1：Methionine cold labeling, external propionating derivatization reaction and proteolysis

1.1 methionine cold labelings

Cell used in the present invention, including HeLa cells (cervical cancer tumer line), K562 cell (chronic myelogenous leukemias Cell), SW620 cells (colon cancer cell), A549 cells (lung carcinoma cell) and SMM7721 cells (HCC), culture exists (Life Technologies, CA) is carried out in RPMI or DMEM culture mediumsMethionine cold labeling.It is main in culture medium To include following composition：¹²CH₃- methionine or¹³CD₃- methionine (Sigma-Aldrich, MO), 10% dialysis hyclone (v/v) (dialyzed FBS) (Life Technologies, CA) and 1 × antibiotic (Thermo Scientific, MA) with And the nutrient of other suitable cell growths.Cell is at 37 DEG C and 5%CO in culture medium₂In carry out grown cultures.

Cold labeling it is critical only that labeling effciency will reach more than 97%, and have Mass Spectrometer Method to be confirmed. Often divide a generation 50% according to cell¹²CH₃- quilt¹³CD₃- calculating (be under physiological status¹²CH₃-), when cell continuously divides 6 After generation, in vivo more than 99%¹²CH₃- quilt¹³CD₃- replace, so that the cold labeling efficiency needed for meeting theory. It is parallel carry out " light " (¹²CH₃- Met) or " weight " (¹³CD₃- Met) stable isotope cell marking during, when " weight " (¹³CD₃- Met) stable isotope mark reach requirement after, " light " (¹²CH₃- Met) or " weight " (¹³CD₃- Met) stable isotope The cell of mark needs to continue to cultivate the cell quantity (10 until required⁸) enough albumen can be extracted for follow-up reality Test.After cell culture is required to satisfaction, the Qualitative Identification of lysine monomethyl modification in cell is such as carried out, only collect " weight " (¹³CD₃- Met) mark cell after carry out total protein extraction.The quantitative analysis of lysine monomethyl modification in cell is such as carried out, Then " light " (¹²CH₃- Met) or " weight " (¹³CD₃- Met) mark cell collect after mixed in equal amounts carry out the extraction of albumen.

The extraction of total protein in 1.2 mark cells

The cell for cultivating mark is washed 3 times before collection with the phosphate buffer (PBS) of precooling, then in the cracking of precooling Cell lysis (8M in buffer solutionUreaIt is dissolved in 2:1 0.1M NH₄HCO₃Buffer solution (PH=8) and 0.1M NaHCO₃Solution；1x Protease inhibitors (v/v)), half an hour is finally incubated on ice.Centrifugation (20000x g) removes cell fragment, retains supernatant, And determine the content of total protein in supernatant fluid.

The external propionating derivatization reaction and proteolysis of 1.3 total proteins

The specific step of the external propionating derivatization reaction of total protein is as follows：

1) the Protein Extraction supernatant of each cell line in 1.2 sections is taken, wherein each supernatant contains the total eggs of 20mg It is white to prepare propionating reaction；

2) it is vortexed and mixes to 200 μ L propionic andydrides are added in supernatant fluid of the collection rich in protein immediately, adds appropriate body Long-pending 2M NaOH, pH is adjusted to 8.0, room temperature reaction 1 hour.

3) repeat step 2 is once.Rejoin in solution of the 200 μ L propionic andydrides in step 2, mix, add appropriate body Long-pending 2M NaOH, adjust pH to 8.0, room temperature reaction 2 hours.

4) after reaction connects beam, 10 μ L monoethanolamines (ethanolamine) are subsequently added, room temperature 30 minutes terminates step 3 Propionating reaction.

5) in the solution in step 4, trichloroacetic acid (TCA) to final concentration of 20% (v/v) is added dropwise over, it is light on shaking table Shake, 4 DEG C of precipitates overnights.

6) second day 16,000X g, 4 DEG C of centrifugation 10min, abandon supernatant.

7) the resuspended albumen precipitation of acetone of 4 DEG C of precoolings, then 16000X g, 4 DEG C of centrifugation 10min are added, supernatant is abandoned.

8) repeat step 7 is twice.

9) 2mL100mM ammonium hydrogen carbonate, the albumen precipitation in the resuspended steps 8 of pH=8 are added.

10) by pancreatin and the substrate 1 of albumen precipitation:The ratio of 50 (w/w), adds appropriate pancreatin in above-mentioned protein solution In, digested overnight in 37 DEG C of waters bath with thermostatic control.

11) second day, centrifugation added dithiothreitol (DTT) (DTT) to final concentration of 5mM, and 30 are reacted in 55 DEG C of waters bath with thermostatic control Minute.

12) it is centrifuged, adds the iodoacetamide aqueous solution of Fresh to final concentration of 15mM, room temperature lucifuge reacts 30min.

13) after reaction terminates, centrifugation adds 0.72M cysteines, to final concentration 30mM, room temperature reaction 30min.

14) pancreas enzyme-to-substrate 1 is pressed again:The ratio of 100 (w/w), adds appropriate pancreatin, and 37 DEG C of waters bath with thermostatic control are digested 3 hours Afterwards,¹⁸C post desalinations, with HPLC isolated polypeptide components.

Examples of implementation 2：Polypeptide component based on HPLC is separated

Before affinity purification, the pancreatin peptide of the propionating derivatization from each sample obtained by examples of implementation 1 Duan Shouxian is separated by HPLC.The method of separation is as follows：This separation uses Varian SD1LC (Agilent Technologies, CA) and Xbridge C18 (19X150cm；Waters, MA), using 2 to 40% buffer B (10mM Ammoniacal liquor or 80%ACN, pH8.5), gradient is set to 70 minutes, is eluted with flow velocity 10ml/min.Sample is collected Deng the time, is received altogether Collect 60 components (about 10ml/ pipes), it is 20 components to be then combined with.Each component of all 20 components is dense with vacuum respectively Contracting instrument (ThermoFisher) is drained.

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 glucoresins (Life Technologies, CA) after pre-cooled phosphate buffer (PBS) washs three times, and 4mg lysine propionyl methylate it is general anti- Body (in the phosphate buffer of 2ml precoolings) is incubated 4 hours under the conditions of 4 DEG C；After 500x g centrifugations 30s, Antibody-protein A fine jades Lipolysaccharide resin washs three uncombined antibody of removal with the phosphate buffer of precooling, be prepared into lysine propionyl methylate it is general Antibody coupling resin.

The affine enrichment of modified polypeptide：The enzymolysis polypeptide (2mg) obtained by embodiment 2 is dissolved in the NETN of 200ul precoolings Buffer solution (100mM NaCl, 1mM EDTA, 20mM Tris pH8.0and0.5% (w/v) NP-40), is subsequently adding 20ul and relies Propylhomoserin propionyl methylates ubiquitin antibody binding resin, night incubation under the conditions of 4 DEG C.500x g are centrifuged 30s, affine enrichment polypeptide-anti- After body-Protein-A Sepharose glucoresin washs three times with NETN buffer solutions, then washed twice with ddH2O.With reference in Antibody-protein A fine jades Affine enrichment polypeptide on lipolysaccharide resin is eluted with 0.1% trifluoroacetic acid (TFA) of 100ul, totally three times.The wash-out that mixing is three times Liquid, is used for follow-up mass spectral analysis after being drained with vacuum concentration instrument.

Examples of implementation 4：HPLC-MS/MS is analyzed and database retrieval protein sequence

Elute the ionization of peptide fragment：The propionyl being enriched in examples of implementation 3 is methylated peptide fragment (20 peptide fragments of component) 3 μ l HPLC buffer As (0.1% aqueous formic acid, v/v) are dissolved in respectively and then sequentially enter capillary RPLC trapping columns (100 μ M internal diameters x2cm, Luna C18 are filled, 5 μm, apertureChinese enlightening horse) and Autosampler that maximum pressure is 250 handkerchiefs, Buffer solution is 100% buffer A (HPLC grades of the 0.1%FA aqueous solution).After sample introduction and wash-out terminate, peptide fragment is transferred to filling C18 resins (3- μm of granular size,Aperture, Chinese enlightening horse) analytical column (10cm is long, 75 μm of ID), and analytical column is connected Connect EASY-nLC1000HPLC systems (Thermo Fisher Scientific Inc, MA).

Mass spectral analysis：After the peptide fragment for eluting is ionized, LTQ Orbitrap are then imported into by nano-spray In Elite mass spectrographs (Thermo Fisher Scientific Inc, MA).With R=24, the resolution ratio of 000 and m/z400 is entered Row gamut scanning of the mass spectrum (from m/z300 to m/z2,000).10 kinds of ions of highest abundance are once separated in linear ion hydrazine Out, by collision and then division (CID), the normalized energy is 35%.The exclusion unrelated with data was repeated at intervals of 30 seconds 2 are counted as, window are excluded and is arranged on+2Da and -1Da.The HPLC-MS/MS data of acquisition use Mascot (v2.3, Matrix Science, UK) analysis.The extract_msn.exe Software Creates that peak list passes through Thermo Fisher.In Mascot analyses Parent ion quality error is set to ± 10 ppm, and fragment masses error is set to ± 0.6Da.By the data obtained in UniProt Searched in Human (88,817 sequences) database, and search parameter is done into following setting：Fixed modification is set to cysteine Residue urea methylates, and the variable of low quality peptide fragment is modified to¹³CD₃- methionine,¹³CD₃- methionine oxidation, lysine are propionating (lysine+56.0262), lysine propionyl¹³CD₃- methylate (lysine+74.0640) and lysine propionyl-methyl-methyl Change (lysine+70.0418).All mass spectrometric datas are using the mascot ionic fractions filtering higher than 20 and manual verification.

Examples of implementation 5：The purifying of CDCL1 albumen in K562 cells

Will¹³CD₃- methionine weight target K562 cells be dissolved in 1x NETN buffer solutions (100mM NaCl, 1mM EDTA, 20mM Tris pH8.0 and 0.5%w/v, NP-40).12,000 × g is centrifuged and goes out for 10 minutes cell homogenates under the conditions of 4 DEG C Cell fragment.

4 DEG C of anti-CDC5L antibody (Santa Cruz, Texas) is added in lysate overnight, albumin A/G agar is subsequently adding Glucoresin (SantaCruz, Texas) is incubated 4 hours at 4 DEG C.After washing non-specific binding albumen, immunoprecipitate is in loading Separated on 4-12%SDS-PAGE (Life Technologies, CA) after thermal cracking in buffer solution.Destination protein is corresponding Adhesive tape cuts and in film dosim, peptide hydrolysis then is dissolved in into 0.1%TFA buffer solutions and examples of implementation 4 are imported into Analyzed in HPLC-MS/MS mass spectrums.

Examples of implementation 6：The preparation of total protein in hepatoma sample

Liver cancer tissue comes from the liver cancer patient performed the operation in Zhong Shan hospitals (China, Shanghai), and will study interior Appearance is informed and patient or donation people.The flesh tissue for cutting is before being further processed by liquid nitrogen quick freeze.Before extracting albumen, Liver organization is shredded rapidly using surgical scissors and uses the phosphate buffer (PBS) of precooling to wash away blood.It is placed in the PBS of precooling In tissue stirred using agitator, connective tissue filter is filtered in (70 μM of apertures).It is thin by the way that acquisition liver cancer is centrifuged Born of the same parents, and cell culture and mark are carried out by the method that 1.1 in embodiment 1 are saved, cultivated on cell culture medium and be used in combination¹²CH₂- methionine or¹³CD₂- methionine is marked.Enter by the method for the 1.2 of examples of implementation 1 and 1.3 sections after cell marking The extraction of row total protein and propionating reaction is carried out, while also decomposed with enzyme in the same manner, using chromatography point The degree of propionating reaction is analysed, is as a result shown, in the protein of these cells, only few peptide does not carry out propionating anti- Should, all of albumen of essence has all carried out propionating reaction.

Examples of implementation 7：Preparation for identifying the ubiquitin antibody that the third lysine propionyl methylates

The reagent used in the embodiment of the present invention such as following table：

Table 1

Various solution formulas are as follows in the embodiment of the present invention：

Table 2

The preparation of ubiquitin antibody

1st, the synthesis of the synthesis (Lys (me-prop)-OH) of the propionating lysine small molecule of monomethyl.

Synthetic route (Fig. 1 D, wherein NHFMor are blocking group)：

1) 2-1 dichloromethane (DCM) dissolves, and brand-new HCl gases is continually fed under normal temperature after 1.5 hours, under strong acid often Temperature continues to react 5 hours, and TLC confirms that reaction terminates；Sticky solid is obtained after being spin-dried for, is then added silica gel mixed sample to carry out column chromatography and is obtained 2-2；

2) 2-2 adds acetone solution, is adding NaHCO₃It is stirred at room temperature after the aqueous solution 2 hours and (is sufficiently stirred for, removes raw material The HCl of middle combination), the acetone soln dissolved with propionic andydride is slowly added under ice-water bath, react 1 hour；Reaction terminates rear 2N hydrochloric acid PH3.0 or so is adjusted, adds DCM to be extracted, taken organic phase and be spin-dried for obtaining sticky solid, plus silica gel carries out column chromatography and obtains 2-3；

3) 2-3 adds piperidines, stirring at normal temperature after adding DMF (DMF) and tetrahydrofuran (THF) dissolving Overnight, TLC confirms that reaction terminates；Oil pump obtains mix products after subtracting steaming solvent evaporated.Plus silica gel column chromatography purifies to obtain esterification products； Hydrogenation sodium oxide molybdena, equivalent water and methanol system are hydrolyzed, and are reacted 2 hours under ice-water bath, and 2N is used after esterification products complete hydrolysis HCl is adjusted to pH3.0.Solvent is spin-dried for, ethanol lysate is added, filtrate is spin-dried for after filtering, obtain final product Lys (me- prop)-OH。

2nd, prepared by the modified lysine small molecule holoantigen of activation

1) appropriate KLH is dissolved in 1mL activation buffers, adds the EDC of 3.2mg, makes EDC final concentrations 2mM；

2) 1.1mg Sulfo-NHS are added in reaction solution 1；

3) reaction solution 1 and 2, room temperature reaction 15 minutes are fully mixed；

4) 1.4 μ L mercaptoethanols are added to terminate EDC reactions；

5) appropriate Lys (me-prop) is dissolved in activation buffer, is subsequently added in the KLH protein solutions after activation, adjustment PH to 7.4；

6) reaction solution is sufficiently mixed, is reacted 2 hours at room temperature；

7) 50mM Tris terminating reactions are added.

3rd, animal immune program

1) 8 week old new zealand white rabbit, subcutaneous multi-point injection；

2) it is immune for the first time：The small molecule immune for taking the KLH couplings that 500 μ L concentration are 0.8mg/ml is former complete with same volume Freund adjuvant mixing and emulsifying, dorsal sc multiple spot is immunized；

3) after 3 weeks, immunogene and the incomplete freund adjuvant mixing and emulsifying of same volume that 500 μ L concentration are 0.4mg/ml are taken Afterwards, dorsal sc multi-point injection；

4) every 2 weeks, by the dosage of step 3 with method booster immunization once；

5) take 1ml serum within 10 days after the 4th is immune, serum titer is detected with ELISA method.If serum titer is more than 30,000, Pass through Culling heart blood.If serum titer is unqualified, continue repeat step 4, until serum titer is more than 30,000.

4th, polyclonal antibody purification

Protein A prepurifications：

1) serum 10000r/min of the titre more than 30,000 is centrifuged 10 minutes, draws 0.45 μm of filtering with microporous membrane of supernatant；

2) 3ml Protein A resins equilibrium at room temperature 1 hour, the 4 times of phosphate buffer of column volume (PBS) cleaning trees Fat；

3) filter after serum 20ml be loaded in the pillar containing Protein A resins, and with Protein A pillars room After temperature is incubated 90min, 15min is stood；

4) washed 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) 5 times of wash-out Buffer wash-outs of column volume, are subsequently added neutralization buffer, and 4 DEG C of PBSs are overnight；

6) ultrafiltration IgG to concentration be 5-10mg/ml.

It is prepared by antigen polypeptide coupling pillar

1) equilibrium at room temperature SulfoLink Coupling Resin1 hours, after mixing draw 5ml in chromatograph void column in, 4 times The coupling buffer cleaning of column volume is twice；

2) 5mg antigen polypeptides are weighed, is dissolved in coupling buffer respectively, add Resin layers of SulfoLink Coupling Mixed in analysis post post, be incubated at room temperature 15 minutes；

3) 30 minutes are stood, the coupling buffer of 15 times of column volumes washes pillar；

4) 1 times of column volume Block buffer is added, after being incubated at room temperature 15 minutes, 6 times of column volume cleaning buffer solution B clean posts Son；

5) obtain respectively and be coupled pillar and the coupling pillar of polypeptide 2 containing polypeptide 1.

Antigen polypeptide affinity purification

IgG after Protein A purification ultrafiltrations is added into antigen polypeptide coupling pillar, is incubated at room temperature 2 hours；Give up After efflux, respectively with 5 times of cleaning buffer solutions and 10 times of PBS pillars of column volume of column volume, then according to the following steps Movable antibody purification：

1) 4 times of column volume elution buffer wash-outs；

2) neutralization buffer is added in eluent, in bag filter in 4 DEG C of PBSs overnight；

3) ultrafiltration IgG to concentration be 5-10mg/ml；

4) IgG after above-mentioned ultrafiltration is added to polypeptide 2 and is coupled in pillar, be incubated at room temperature 30 minutes, collect efflux, i.e., It is the antibody for having purified.

5th, Dot blot (Dot Blot) detection：

1) by various polypeptide (K_pr+meRepresent lysine propionyl methylated polypeptides storehouse, K_propRepresent the propionating polypeptide of lysine Storehouse, 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_crRepresentative represents lysine crotons and is acylated peptide library) it is soluble in water, It is configured to the polypeptide solution that initial concentration is 100ng/ μ L；

2) pvdf membrane of suitable size is cut, absolute methanol is processed about 40 seconds, is rinsed in deionized water 3 times, done naturally Dry 2 minutes；

3) by initial concentration for the polypeptide solution of 100ng/ μ L is further diluted to 20ng/ μ L and 4ng/ μ L respectively.Then Point sample, 1 μ l/ points, are put into six orifice plates after drying 10 minutes successively；

4) 5% skimmed milk power room temperature is closed 60 minutes, and TBST washing lotions are rinsed twice, 5 minutes/time；

5) antibody is diluted with 5% skimmed milk power, is incubated at room temperature 1 hour, rinsed three times with TBST washing lotions, 10 minutes/time；

6) goat-anti rabbit HRP labelled antibodies are diluted with 5% skimmed milk power, room temperature 45 minutes, TBST washing lotions are rinsed three times, 10 points Clock/time；

7) chemiluminescence nitrite ion is uniformly laid on film, is incubated post-exposure in 5 minutes, as a result see Fig. 1 E.

Dot blot detection shows that being used as antigen by the propionating lysine small molecule of monomethylation of present invention design obtains The polyclonal antibody for obtaining can be with specific recognition lysine methyl-prop acyl polypeptide storehouse, the propionating GG polypeptides of lysine methyl, list The propionating lysine small molecule that methylates and KLH coupling the propionating lysine small molecule of monomethylation, but simultaneously nonrecognition other The polypeptide (Fig. 1 E) of type polylysine modification, thus illustrates the good specificity of antibody.The monomethylation developed is propionating Lysine polyclonal antibody is capable of identify that the lysine methyl-prop acyl polypeptide storehouse of 4 nanograms, but nonrecognition structure it is similar until 100 nanograms other modified lysine peptide libraries, this had both shown the sensitivity of developed antibody, had further showed that this is more The specificity of clonal antibody.

Result 1

By taking HeLa cells as an example, identify in HeLa cell lines whether bad ammonia according to the method for above-mentioned fact Example 1-4 The modification of sour monomethylation and the site modified are, it is necessary to general¹³CD₃Methionine labeling method is combined with SILAC methods.It is real When testing room, by HeLa cell lines, parallel being incubated at contains respectively¹²CH₃- methionine and¹³CD₃In methionine culture medium.Cell mark Note is complete and when reaching the amount of needs, cell extraction total protein is collected respectively, then by same amount of two kinds of crack proteins External propionating derivative reaction is carried out after mixing, pancreatin digestion produces enzymolysis polypeptide, produced with preparation HPLC isolated polypeptide 20 polypeptide fractions.Separate each polypeptide fractions lysine propionyl methylate ubiquitin antibody enrichment, then use nano- HPLC/MS/MS analyzes (specific analytical method is shown in examples of implementation 4).

During mass spectral analysis, occur propionyl methylate modification polypeptide because in methyl group hydrogen ion be set to carbon ion Change, it will produce the mass shift (Fig. 3 A) of 4.002 dalton.It is all to be searched for by Mascot to ensure the accuracy of analysis MS/MS collection of illustrative plates further across artificial nucleus couple.183 confidence levels high should be identified from HeLa cells in this way Hydroxypropyl methyl modified polypeptide, so as to indirectly demonstrate the lysine that there are 183 monomethylation modifications in HeLa cells Presence.By artificial nucleus to initial data, wherein, 180 hydroxypropyl methyl modified polypeptides have a pair of precursor ions, correspondence 190 decorating sites that methylate (Fig. 3 C) in 162 kinds of albumen.Only two polypeptides are containing only " weight (heavy) " ion, one Polypeptide is containing only " light (light) ion.

This is absolutely proved¹³CD₃Replacing the method for methyl group is used for monomethylation site atlas analysis and rejects false positive Result is reliable.Also demonstrate that this method for identifying that monomethyl modified polypeptide is reliable.

Result 2

For application¹³CD₃Methionine labeling method have detected K562 cells (chronic myelogenous leukemia cell), SW620 Cell (colon cancer cell), A549 cells (lung carcinoma cell) and SMM7721 cells (HCC)) etc. other 4 kinds of people sources tumour Lysine monomethylation modification in cell, step and condition of the specific steps as described by examples of implementation 1-4.Wherein, polypeptide Identification uses MASCOT algorithms and 1% false positive rate, record of the MASCOT score values less than 20 is rejected, using strict standard, people Work confirms each collection of illustrative plates (as previously described)²².448 monomethyl polypeptides of nonredundancy are identified altogether, in 403 albumen of correspondence 460 sites.In these polypeptides for identifying, 73% MASCOT score values are higher than 30 (Fig. 4 A).It is public by retrieving Uniprot Protein Data Banks, the present invention identifies the most of monomethyl site come and not yet discloses report.

The monomethyl lysine polypeptide and the complete list of albumen identified by the present invention are seen attached list.This shows, uses The method of the present invention can go out those monomethyl lysine polypeptides and albumen for having been identified by other method with precise Identification, Meanwhile, also identify many other monomethyl lysine polypeptides and albumen for occurring that can not be identified in the past.Fig. 7 shows The 29 lysine monomethylation modifying proteins and decorating site identified in five tumor cell lines.

Knowable to the result identified more than, using the method for the present invention, ignorant single first before those can be identified The polylysine modification of baseization modification.For example on the protein substrate of STUB1 gene codes, lazy propylhomoserin of the present invention in the 2nd position (K2) presence of lysine monomethylation modification is detected on, and the modification of the lysine monomethylation in the site is not yet reported.

The lysine monomethylation of core histones is widely studied, thus can be used to as good positive control. Consistent with estimated, the inventive method identifies 12 lysine monomethyl sites of core histones altogether.Except identifying Outside widely studied histone H 3 K4, H3K9, H3K27, H3K36, H3K79 and the several methylation sites of histone H 4 K20, also reflect Make the histone H 3 K18 monomethylations site almost do not studied.Due to all containing most of these lists in this five kinds of cells Methylate decorating site, illustrates that the method for this detection lysine monomethylation modification is reliable and stable.Additionally, present invention side Method also identifies some new decorating sites from many known monomethylation modified proteins.As in WIZ albumen, except identifying Outside the K976 sites reported, the present invention also identifies the new monomethylation sites (Fig. 7) of K1321 and two kinds of K1463.

Result 3

In order to verify the reliability of the inventive method, with¹³CD₃As a example by the K562 cells of-Met marks, by of the invention Method, identifies and there is monomethylation modification on the K114 sites of the CDC5L albumen of K562 cells.The total protein of K562 cells After digestion was gone forward side by side by external propionating reaction, a sequence for polypeptide of acquisition is LANTQGK^* _pr+meK_prAK_prR (K^* _pr+meRepresent propionating monomethylation lysine, K_prExpression is also connected with propionyl group on lysine).Retouched from above State it is recognised that before the peptide sequence does not carry out external propionating reaction, the polypeptide sequence should in biological cell or tissue For：LANTQGK_me ^*KAKR(K^*Expression there occurs that monomethylation is modified on the site, i.e. 114 sites).In order to verify the knot By inventor is to artificial synthesized LANTQGK^* _pr+meK_prAK_prR has carried out Tandem Mass Spectrometry Analysis.Result shows, the synthesis polypeptide With the LANTQGK identified from cell^* _pr+meK_prAK_prR polypeptides have bis- grades of spectrograms of duplicate MS/MS, but corresponding B-, y- ion on have the mass shift (Fig. 5 A) of 4Da.In order to further verify that the site truly there occurs in cell body Lysine monomethylation is modified, and the present invention identifies lysine list first after additionally using after cell marking CDC5L protein purifications in vivo The Tandem Mass Spectrometry Analysis with artificial synthetic polypeptide of baseization modification.Identified in the CDC5L albumen of purifyingPolypeptide.To artificial synthesizedThe mass spectral analysis of polypeptide shows that this is more Bis- grades of spectrograms of MS/MS of peptide are arrived with purifying CDC5L Identification of Fusion ProteinUnanimously, only because¹³CD₃- Being marked on corresponding b-, y- ion has the mass shift of 4Da, thus demonstrates the lysine list identified in CDC5L albumen Methylate the reliability of modification.

Identification to the lysine monomethyl modification in the liver cancer tissue of people further demonstrates the reliability of the inventive method Property.Data with the lysine monomethylation modification atural object identified in above-mentioned five kinds of cells compare, the identification in liver cancer tissue To 29 monomethylation modified polypeptides in 32 monomethylation sites all contain in this 5 kinds of cells, including 20 new Methylation sites and 6 lysine monomethylation decorating sites of histone H 3 (Fig. 4 B, Fig. 7).

Discuss

Although the protein science strategy based on affine enrichment has been used for many methyl of identification of cell lysate recently Change peptide fragment, however it remains the small physicochemical property that challenge, particularly monomethylation modification cause makes it be difficult to conventional bad ammonia The method of sour monomethylation antibody enrichment is identified.Using the method for the present invention, inventor is identified in 403 substrate proteins 460 lysine monomethylation sites, the maximum group this represent current lysine monomethylation learns data.The result is clearly Confirm the validity and reliability of the method for the present invention.

Although highly effective for lysine monomethylation, our method is not particularly suited for lysine di-methylation and three The group level detection for methylating, because the amino acid residue of both modifications can not be with propionyl anhydride reactant.We also use¹³CD₃- Methionine labelling strategies detect the accuracy of monomethylation peptide fragment to improve.But this method is not directly applicable clinic Tissue samples, the reliability of polypeptide identification can also alternative solve, such as strict manual verification and synthesis polypeptide MS/MS is analyzed.In recent years, the biological relevance of protein lysine monomethylation causes the extensive concern of research institution.Egg White lysine methylates enzyme may target spot by the medicine as various disease.Additionally, some lysine Regulation by Methylation The inhibitor of enzyme is at present in clinical assessment.However, the existing knowledge of lysine monomethylation is confined to a limited number of eggs In vain, such as histone and p53, so as to limit the modification of lysine monomethylation in basic medical research or even course of drug development Application.Our research provide not only a kind of method of effective lysine monomethylation substrate high throughput identification, and Extend the known directory of lysine monomethylation albumen.By proteomics method described in the invention and the enzyme that methylates The genetic manipulation of expression is combined, and is methylated under the substrate, the disease conditions that can operate with identification transmethylase and demethyl enzyme The dynamic analysis of substrate.Additionally, lysine monomethylation modification group provided by the present invention will be further biological study Meaningful starting point, realizes the functional characterization description of lysine monomethylation albumen with disease specific lysine monomethylation on the way The parsing in footpath.

Claims

1. the method for monomethylation modification there is on a kind of ε-amido for identifying lysine residue in substrate, the method includes：

1) external nitrogen acylation derivative reaction is used, it is acyl methyl that the lysine residue monomethylation ε on substrate-amido is derived Change ε-amido；

2) acyl prepared using the specificity affine enrichment acyl of lysine ubiquitin antibody that methylates is methylated the peptide fragment of modification；

3) acyl for analyzing and identifying the affine enrichment of antibody using mass spectrum and data methylate modification site and polypeptide；

Wherein, described acyl methylates lysine ubiquitin antibody for propionyl methylates lysine ubiquitin antibody, and the method also includes preparing Specific propionyl methylates lysine ubiquitin antibody, described to prepare specific propionyl and methylate lysine ubiquitin antibody step bag Include following steps：

Synthetic route is：

Wherein, NHFMor is blocking group；

What is synthesized comprises the following steps that：

(1), 2-1 dichloromethane (DCM) dissolves, and brand-new HCl gases is continually fed under normal temperature after 1.5 hours, normal temperature under strong acid Continue to react 5 hours, TLC confirms that reaction terminates；Sticky solid is obtained after being spin-dried for, is then added silica gel mixed sample to carry out column chromatography and is obtained 2- 2；

(2), 2-2 adds acetone solution, is adding NaHCO₃It is stirred at room temperature after the aqueous solution 2 hours, allows it to be sufficiently stirred for, is removed former The HCl combined in material；The acetone soln dissolved with propionic andydride is slowly added under ice-water bath, is reacted 1 hour；Reaction terminates rear 2N hydrochloric acid PH 3.0 or so is adjusted, adds DCM to be extracted, taken organic phase and be spin-dried for obtaining sticky solid, plus silica gel carries out column chromatography and obtains 2-3；

(3), 2-3 adds piperidines, stirring at normal temperature mistake after adding DMF (DMF) and tetrahydrofuran (THF) dissolving Night, TLC confirms that reaction terminates；Oil pump obtains mix products after subtracting steaming solvent evaporated；Plus silica gel column chromatography purifies to obtain esterification products；Plus NaOH, equivalent water and methanol system are hydrolyzed, and are reacted 2 hours under ice-water bath, and 2N is used after esterification products complete hydrolysis HCl is adjusted to pH 3.0；Solvent is spin-dried for, ethanol lysate is added, filtrate is spin-dried for after filtering, obtain the propionating single first of final product Base lysine small molecule, the preparation of small molecule holoantigen, Ran Houyong are carried out to obtaining propionating monomethyl lysine small molecule again The antigen for preparing carries out immune animal, and the serum obtained to animal is immunized carries out the purifying of antibody, final to obtain special The propionyl of property methylates lysine ubiquitin antibody.

2. method according to claim 1, substrate lysine residue monomethylation ε-amido derive for propionyl methylate ε- Amido.

3. method according to claim 2, is methylated the position of modification using the propionyl lysine ubiquitin antibody that methylates to propionyl Point is enriched with.

4. method according to claim 1, nitrogen acylation modification is carried out to substrate lysine residue monomethylation ε-amido, is repaiied Adorn the alkyl or aromatic radical of the total number of carbon atoms less than 8 of group.

5. method according to claim 4, described substrate is polypeptide or albumen, and acylation reaction can disappear in protease Carried out before or after changing substrate.

6. method according to claim 5, lysine ubiquitin antibody is methylated to acyl methyl using corresponding substrate protein acyl The site for changing modification is enriched with.

7. the bottom of monomethylation modification there is in method according to claim 6, the lysine residue to be identified ε-amido Thing is to be extracted from the albumen or polypeptide obtained in any cell, tissue or body fluid.

8. method according to claim 7, the substrate protein white matter for being extracted from any cell, tissue or body fluid can be carried out in vitro Chemical derivatization reaction；Acylating reagent is the acylating reagent with stable isotope.

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.

10. method according to claim 9, modifies lysine monomethylation the quantitative analysis of substrate, can select steady Determine isotope acylating reagent and analyze three groups of different lysine monomethylation modification substrates；Three groups of described different methyl of analysis Change modification substrate, it can be carried out¹²C₆H₁₀O₃,¹³C₆H₁₀O₃With¹²C₆D₁₀O₃Mark respectively.

11. methods according to claim 10, cell is that can carry out the cold labeling based on cell culture (SILAC) cell of immortality passage.

12. methods according to claim 11, are lysine monomethylation modification substrate in identification of cell, and cell can be cultivated In the SILAC culture mediums for being added with stable isotope amino acid, described isotope amino acid includes¹²C- arginine or¹³C- Arginine,¹²C- lysines or¹³C- lysines,¹²CH₃- methionine or¹³CD_3-Methionine.

13. methods according to claim 12, cell culture is containing¹²CH₃- methionine or¹²CD_3-Methionine In SILAC culture mediums, the Qualitative Identification of lysine monomethylation modification can be both carried out, can also carry out lysine monomethylation modification Quantitative analysis.

14. methods according to claim 13, be extracted from the protein of any cell, tissue or body fluid can carry out it is external Chemical derivatization reacts, wherein, described derivative reaction is that the acylation reaction on protein lysine residues ε-amido is Propionating derivative reaction.

The albumen of 15. methods according to claim 14, substrate protein or external propionyl derivatization carries out protease and disappears Change and produce peptide hydrolysis.

16. methods according to claim 15, the enzymolysis polypeptide of generation can further with alkaline high performance liquid chromatography point From.

17. methods according to claim 1, propionyl methylates the chemical crosslinking of lysine ubiquitin antibody, covalent bond or non-co- Valency is combined in any matrix for not destroying antibody compatibility, wherein, described matrix is Non-covalent binding in Protein A sepharose tree Antibody coupling resin is prepared on fat.

18. methods according to claim 1, the affine enrichment of antibody is judged by the result of Mass Spectrometric Identification and MASS SPECTRAL DATA ANALYSIS Methylated modification with the presence or absence of propionyl on the lysine ε-amido of peptide fragment, including decorating site, modified polypeptide sequence and modification The corresponding albumen of polypeptide.

19. profits require the method described in 18, and mass spectral analysis may include that Matrix Assisted Laser Desorption time-of-flight mass spectrometry is analyzed, nanoliter Liquid phase Tandem Mass Spectrometry Analysis or three-level mass spectral analysis.