CN103776909A - Identification method of protein ubiquitination modification sites - Google Patents
Identification method of protein ubiquitination modification sites Download PDFInfo
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- CN103776909A CN103776909A CN201210398526.0A CN201210398526A CN103776909A CN 103776909 A CN103776909 A CN 103776909A CN 201210398526 A CN201210398526 A CN 201210398526A CN 103776909 A CN103776909 A CN 103776909A
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Abstract
The invention relates to a protein ubiquitination modification site identification method, which comprises the steps of firstly carrying out enzymolysis on purified ubiquitination-like modified protein, enriching a ubiquitination-like modified enzyme digestion peptide segment through a specific ubiquitination-like antibody, then protecting non-modified lysine amino residues in the enriched peptide segment by using a chemical marker, exposing modified lysine on a substrate peptide segment sequence through deubiquitinating enzyme catalysis reaction, analyzing the peptide segment sequence through a liquid chromatography tandem mass spectrometer, and obtaining the chemically modified peptide segment sequence through matching of protein mass spectrum data analysis software, wherein the lysine site containing free amino in the sequence is the ubiquitination-like modification site. Compared with the site obtaining mode of the current common gene mutation method, the invention has the advantage that the protein ubiquitination modification site information can be quickly, efficiently and accurately obtained without complicated gene mutation technical means.
Description
Technical field
The present invention relates to proteomics field, more particularly, relate to evaluation and the application in protein-based ubiquitination site.
Background technology
In cell, have multiple proteins posttranslational modification mode, as phosphorylation, acetylation, ubiquitination, methylate etc., the biological function that they are all brought into normal play to protein plays an important role.Wherein ubiquitination (ubiquitylation) is one of important way of mediating protein degraded, by by 76 amino acid whose ubiquitin (ubiquitin, Ub) be attached on target protein and form poly ubiquitin chain, identified by proteasome, cause protein degradation.Several ubiquitin-like albumen (ubiquitin-likeproteins in the last few years, Ubls) be found successively, their structures and ubiquitin are similar, in cell, have wide variety of functions, SUMO (small ubiquitin-related modifier) is exactly one of important member wherein.SUMO is the protein families of a class high conservative, and first member SMT3 of this family was found in saccharomyces cerevisiae first in nineteen ninety-five, thought that at that time it has the mitosis of participation and maiotic function.Along with further deeply finding of research, SUMOization (sumoylation) function is far not limited to this.SUMO can with comprise androgen receptor (androgen receptor; AR), I κ B α, c-Jun, histon deacetylase (HDAC) (histone deacetylases; HDACs) and p53 in interior multiple proteins combination; participate in endocellular metabolism approach more widely, all played an important role in many-sides such as transcriptional regulatory, DNA reparation, nuclear translocation, signal transduction and cell cycle regulatings.
Having the SUMO of rhetorical function, is the protein family that a class is extensively present in the high conservative in eukaryotic cells, all has expression in the vertebrate cells of budding yeast, nematode, fruit bat and cultivation.Four kinds of different SUMO albumen of human gene group coding, be respectively: SUMO-1 (claims again PIC1, UBL1, sentrin, GMP1 or SMT3), SUMO-2 (claim not only SMT3A or sentrin3), SUMO-3 (but also claiming SMT3B or sertrin2) and SUMO-4, wherein SUMO-1, SUMO2 and SUMO3 all have expression in various tissues, and SUMO-4 mainly expresses in kidney, lymph node and spleen.Wherein SUMO-2,3 have 97% homology, and with the homology of SUMO-1 only 50%.Sequence alignment result shows: all SUMO albumen all has the fracture of the two Gly of conservative ubiquitin domain and C end/is connected site, and N holds elongated area to be rich in charge residue, Gly and Pro, for special protein-protein interaction provides architecture basics.
SUMOization is dynamic reversibly modified process, all SUMO exist with inactive precursor forms, need be at ubiquitin similar protein processive enzyme (ubiquilin like protein progressing enzyme, or SUMO specific protease (SUMO specific proteases ULPs), SENP) under catalysis, be hydrolyzed C end small peptide to expose GlyGly residue, become ripe SUMO.Similar to ubiquitination, ripe SUMO under ATP exists, through SUMO activating enzymes E1, SUMO desmoenzyme E2, the cascade reaction of SUMO ligase E3, finally its C end Gly residue passes through isopeptide bond coupling with target protein substrate Lys side chain epsilon-amino.Research discovery, ligase E3 has determined substrate specificity.Exceeding 50%SUMO substrate target site is a conserved sequence Ψ KXE (Ψ represents the hydrophobic amino acids such as L, I, V, F, and K represents lysine, and X is arbitrary amino acid, and E represents glutamic acid).Another representative conserved sequence is called the SUMO sequence that phosphorylation relies on, and Ψ KXE is being close to the phosphorylation sequence of P mediation.The reversed reaction process that SUMO modifies is to be completed by one group of SUMO specific protease (SUMO-specific proteases, SENPs).
Concentrate single or protein modified the developing into of minority to utilize at present high throughput protein omics technology from the mutating technology that utilizes in the past to the excavation of SUMOization modifying protein, carry out excavation and the evaluation of extensive SUMOization modifying protein.Compared with the phosphorylation site ten hundreds of with document, the research that identify in the SUMOization site identifying is at present phoenix hair water caltrop.Main cause is as follows: first, due to dynamic and the reversibility of SUMOization modification, in normal cell, the albumen of SUMOization accounts for total protein ratio and is no more than 5%, much albumen is only under particular stimulation condition as cell cycle, nutritional status, heat shock, DNA damage etc. just can be modified by SUMOization, how to be enriched to that enough SUMOization peptide sections are identified and the interference of getting rid of unmodified peptide section is the difficult point of research always; Second, the SUMOization SUMOization modified peptides segment molecule amount after making to be cut by pancreatin enzyme of modifying has remarkable migration, and (SUMO-1 modifies and makes peptide segment molecule amount increase 2136Da, SUMO-2/3 modifies and makes peptide segment molecule amount increase 3552Da), if by the electrically charged deficiency of modified peptides section, specific charge is (400-1800) not within the scope of Mass Spectrometer Method, can not be selected, carry out MS/MS Sequence Identification; The 3rd, the SUMOization peptide section of high molecular is chosen while carrying out collision induced dissociation in one-level mass spectrum, because collision energy is limited, often can not provide enough abundant fragmention for Sequence Identification; The 4th, different from phosphorylation modification, the amino acid sequence of SUMO modification part also can produce collision and dissociate, and makes whole SUMOization peptide section MS/MS mass spectrogram quite complicated.Although attempt to some extent, not yet having at present can commercialization data analysis software, can modify mass spectrographic MS/MS to these SUMO and carry out high flux identification, identifies substrate peptide section sequence and decorating site.Vitro reactions system identifies only a small amount of SUMOization site.Take SUMO sequence specific amino acids to suddenly change and can obtain some site data.
Determine protein s UMOization decorating site not only can illustrate may occur modify conserved amino acid sequence or the specific rule of structural motif, and provide important evidence for protein in the potential function of signal path for studying this type of modification, the biological effect of modifying for clear and definite protein s UMO, further investigation SUMOization is modified at the crucial regulating and controlling effect in Growth of Cells, growth, apoptotic process, significant.
Summary of the invention
The difficulty of identifying for solving ubiquitin-like decorating site, the invention provides a kind of can be fast, the method for the protein-based ubiquitination of precise Identification site evaluation.
Second object of the present invention, is to provide the application of chemical labeling in identify in protein-based ubiquitination site.
In order to address the above problem, the invention provides a kind of protein-based ubiquitination site authentication method, it is characterized in that, comprise the following steps:
(1) the SUMOization albumen of purifying being carried out to enzyme cuts;
(2) specific antibody is to the enrichment of SUMOization modified peptides section;
(3) chemical labeling is protected the lysine amino residue of non-modifiedization in institute's enrichment peptide section;
(4) go ubiquitination enzymic catalytic reaction to expose adorned lysine on substrate peptide section sequence;
(5) liquid chromatography mass combined instrument is analyzed peptide section sequence;
(6) utilize proteomic image data analysis software coupling to obtain by the peptide section sequence of chemical modification, the lysine sites containing free amine group in its sequence is ubiquitin-like decorating site;
Described SUMOization is modified and is comprised SUMO1, and SUMO2 and SUMO3 ubiquitin-likeization are modified.
As a preferred version, the described antibody of step (2) refers to the specific antibody that SUMOization peptide section is modified, and through rabbit anteserum immunity gained, can distinguish specific recognition ubiquitin-like and modify SUMO1, the corresponding specific amino acid sequence of SUMO2 or SUMO 3.
As another preferred version, the described chemical labeling of step (3) is the one in dialkyl group modification or acetylation modification or guanidinated modification.
Described dialkyl groupization is modified and is comprised the steps:
(a) sample is dissolved in pH7.5 buffer solution, adds in the aldehydes solution of fresh configuration, mix;
(b) add the original reagent of going back of fresh configuration, mix, after completion of the reaction with stop bath cancellation reaction;
(c) with the desalination of chromatogram pre-column.
As a preferred version, the described aldehydes chemical structural formula of step (a) is R
1cHO, R
1for the C of iodine replacement
1～C
10alkyl, concentration is between 0.01 – 1M;
The described original reagent of going back of step (b) is the one in sodium borohydride, sodium cyanoborohydride or borine, and concentration is between 0.01 – 1M; Between temperature of reaction 0-60 ℃; Reaction time 1-48 hour; Described stop bath refers to acid solution, is one or more in hydrochloric acid, formic acid, acetic acid or trifluoroacetic acid solution, and concentration is between 0.1-5%;
The described chromatogram pre-column of step (c) is Ziptip desalting column.
Described acetylation modification comprises the steps:
(a) sample is dissolved in pH7.5 buffer solution, adds in the N-acetate substituent solution of fresh configuration, mix;
(b) add the deesterify reaction solution of fresh configuration, mix, with stop bath cancellation reaction;
(c) with the desalination of chromatogram pre-column.
As a preferred version, the described N-acetate substituent solution of step (a) is one or more in N-acetate acetyl chloride, N-acetoxyacetic acid, N-acetate succinimide or N-acetate glycocoll, concentration is between 0.01-1M, between temperature of reaction 25-60 ℃; Reaction time 1-24 hour;
The described deesterify reaction solution of step (b) is oxammonium hydrochloride, Hydroxylamine sulfate, N, one or more in N-diethyl hydroxylamine, hydroxylamine nitriate or phosphatic hydroxylamine, concentration is between 0.01 – 1M, described stop bath is one or more in glutamic acid, glycocoll or leucine, and concentration is between 1-100mM;
The described chromatogram pre-column of step (c) is Ziptip desalting column.
Described guanidinated modification comprises the steps:
(a) by sample dissolution in alkaline buffer solution, add the adjacent Methyl Isourea Sulfate solution of fresh configuration, start chemical reaction;
(b) add acidic buffer solution, adjust pH is to acid cancellation reaction;
(c) with the desalination of chromatogram pre-column.
As a preferred version, the described alkaline buffer solution of step (a) is one or more in NaOH, potassium hydroxide, sodium carbonate or ammoniacal liquor, and concentration is between 1-10M; Temperature of reaction between-20-100 ℃, reaction time 1-24 hour;
The described acidic buffer solution of step (b) is one or more in formic acid, acetic acid, butyric acid, acetic acid or trifluoroacetic acid, and concentration is between 0.01%-20%;
The described chromatogram pre-column of step (c) is Ziptip desalting column.
As another preferred version, go ubiquitination enzymic catalytic reaction to comprise the steps: described in step (4)
(a) sample is dissolved in again to ubiquitination enzyme and enzymic catalytic reaction buffer solution, mixes 37 ℃ of reactions;
(b) chromatogram pre-column desalination.
As a preferred version, go ubiquitination enzyme to refer to SENP described in step (a), comprise SENP1, one or more in SENP2 or SENP3; Described enzymic catalytic reaction solution is to contain phosphoric acid, Tris-HCl or the Hepes-HCl buffer solution of going back original reagent and sodium chloride, the described original reagent of going back is dithiothreitol (DTT) or beta-mercaptoethanol solution, reduction reagent concentration is between 0.001 – 1M, and sodium chloride concentration is between 0.05 – 1M;
The described chromatogram pre-column of step (b) is Ziptip desalting column.
In order to realize second object of the present invention, the invention provides the application of chemical labeling in identify in protein-based ubiquitination site.
As a preferred version, protein-based ubiquitination site is identified and is comprised the steps:
(1) the SUMOization albumen of purifying being carried out to enzyme cuts;
(2) specific antibody is to the enrichment of SUMOization modified peptides section;
(3) chemical labeling is protected the lysine amino residue of non-modifiedization in institute's enrichment peptide section;
(4) go ubiquitination enzymic catalytic reaction to expose adorned lysine on substrate peptide section sequence;
(5) liquid chromatography mass combined instrument is analyzed peptide section sequence;
(6) utilize proteomic image data analysis software coupling to obtain by the peptide section sequence of chemical modification, the lysine sites containing free amine group in its sequence is ubiquitin-like decorating site;
Described SUMOization is modified and is comprised SUMO1, and SUMO2 and SUMO3 ubiquitin-likeization are modified.
As one preferably, described chemical labeling refers to dialkyl group modification, acetylation modification or guanidinated modification.
SUMOization the protein purification of the present invention and SUMOization albumen of purifying is carried out to enzyme be cut to conventional steps, generally comprises following steps:
(a) go back original reagent by adding in protein solution, protein disulfide bond is opened;
(b) add alkylating reagent, lucifuge incubated at room 30min, by albumen mercaptoalkyl;
(c) get 3-10mg albumen, add appropriate proteolytic enzyme, after enzymolysis, digestion, end enzymatic activity;
(d) after vacuum decker concentrate drying powdered, then add 1mL deionized water, repeatedly repeat this process 3-4 time.
The described original reagent of going back of step (a) is recommended as dithiothreitol (DTT) or beta-mercaptoethanol etc.
The described alkylating reagent of step (b) is recommended as R
1(CO) NH
2, wherein: R
1be recommended as the C that iodine replaces
1～C
10alkyl, be preferably iodine replace C
1the alkyl of～C4.
The described proteolytic enzyme of step (c) can be typsin, Lys-C, Arg-C etc.And the reaction ratio of albumen is recommended between 1:50-1:100, depending on enzymatic activity.
To SUMOization modified peptides section, enrichment is the routine operation step that adopts the enrichment of peptide section to step (2) specific antibody, generally comprises following steps:
(a) peptide hydrolysis is dissolved in to antibody enrichment buffer solution, adds antibody, gentle rotation hatched after 12h, washes three times the centrifugal 30s of 12000g with enrichment buffer solution;
(b) the specificity microballon that can identify antibody joins in (a) solution, and gentle rotation hatched 4-8h;
(c) centrifugal 1000g, carefully removes supernatant, 1mL washing buffer solution washing microballon three times;
(d) remove liquid completely, add 150uL elute soln, repeated washing 3 times, collects all supernatants, vacuum drying.
It should be noted that, the described specific antibody that SUMOization peptide section is modified of step (2), through rabbit anteserum immunity gained, its antibody is characterised in that, specific recognition ubiquitin-like be can distinguish and SUMO1, the corresponding specific amino acid sequence of SUMO2 or SUMO3 modified.
The described enrichment buffer solution of step (a) is the buffer solution of pH value 8.0, sodium chloride-containing, sodium ethylene diamine tetracetate and a small amount of surfactant.Buffer solution is recommended as Tris-HCl buffer solution.Sodium chloride concentration is between 0.05-1M, and ethylenediamine tetraacetic acid concentration is between 5-100mM, and surfactant can be Brij35, Triton-X100 etc., and concentration is between 0.005-5%.
The specificity microballon of the antibody identified described in step (b) is recommended as commercial proteinA microballon.
The described washing buffer solution of step (c) can be phosphoric acid, Tris-HCl buffer solution or Hepes-HCl buffer solution.
The described elution buffer solution of step (d) refers to acid solution, can be hydrochloric acid, formic acid, acetic acid, trifluoroacetic acid solution, and concentration is between 0.1-5%.
The present invention's liquid chromatography-mass spectrography used is used in conjunction instrument and refers to receive and rise liquid chromatograph-mass spectrometer, is the conventional detecting instrument using in this area, and recommendation liquid chromatograph is Agilent1200, and recommendation mass spectrum is LTQ-Orbitrap.Instrument parameter is set according to instrument model.
The present invention's data analysis software used refers to the commercial proteomic image data analysis evaluation such as Sequest, Moscot, Protein Discovery software.Search parameter is used in conjunction instrument configuration according to liquid chromatography-mass spectrography and determines, is those skilled in the art's routine operation method.
The invention has the advantages that: (1) the present invention is cut peptide section by the enrichment of specificity ubiquitin-like antibody by the enzyme of modification first in the detection in protein-based ubiquitination site and authentication method; utilize subsequently chemical labeling to protect the free amine group of institute's enrichment peptide section; and through going ubiquitination enzymic catalytic reaction to expose adorned lysine on substrate peptide section sequence; only need within 2-3 days, can complete whole flow process, do not need loaded down with trivial details mutation technique means can obtain fast, efficiently and accurately protein-based ubiquitination site information.(2) can analyze mass spectrum qualification result by commercial goods search software, without writing specially SUMOization peptide section mass spectrophotometry program, a large amount of manpower, material resources and financial resources that having saved develops software expends.(3) method is reliable and stable, and repeatability is good.
Accompanying drawing explanation
Fig. 1 is ubiquitin-like decorating site authentication method schematic flow sheet.
Fig. 2 A is IPTG abduction delivering Rangap-His-SUMO1SDS-PAGE figure; Fig. 2 B is that Western Blot proves target molecular weight region, and albumen Rangap-His-SUMO1 is expressed.
Fig. 3 is albumen Rangap-His-SUMO1 expression and purification chromatogram.
Fig. 4 is the second order ms figure of SUMOization modified peptides section GFQANVFITSLLVQMGLL (ELGMEEEDVIEVYQEQTGG) KSEDK.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique using in following embodiment if no special instructions, is conventional method.Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment: Rangap-His-SUMO1 ubiquitin-like decorating site is identified
1. destination protein purifying
(1) build RanGap fragment and the people source His-SUMO1 co-expression plasmid (hereinafter to be referred as Rangap-His-SUMO1) containing Africa xenopus, little the shaking of 25 μ l bacterium inoculation 25ml LB nutrient culture media, shakes greatly by 1:100 inoculation bacterium liquid to 2 × YT nutrient culture media, 2L/ bacterium, add corresponding microbiotic, 280rpm, 37 ℃, after about 2-3h, detect bacterium liquid OD600, treat that bacterium liquid temp is slightly down to 20 ℃ of left and right of room temperature, add IPTG induction, 180rpm, 25 ℃, 12h.
Collect the mycoprotein after IPTG induction, also collect the bacterium liquid 2OD of equal quantities, examine to dye and detect albumen and whether be induced to express.12%SDS-PAGE examines and dyes as shown in Fig. 2-A, in target molecular weight region, has albumen to be expressed, and proves that by Western Blot this band is that Rangap-His-SUMO1 is as shown in Fig. 2-B.
(2) add lysate, high pressure fragmentation, to no longer thickness of bacterium liquid, ultracentrifugation, 20,000rpm, 1h, 4 ℃.
(3) adopt protein purification instrument to carry out purifying to target protein.Chromatographic column is nickel post.A is 20mM imidazoles mutually, 0.5M sodium chloride, 20mM sodium phosphate; B is 200mM imidazoles mutually, 0.5M sodium chloride, 20mM sodium phosphate.Flow velocity is 5mL/min, adopts 20 minutes gradients, and 20 minutes internal linear gradients are from 0-100%B, 10 times of column volume 100%B subsequently, and 10 times of column volume 100%A rinse chromatographic columns.
Chromatogram when Fig. 3 is protein purification.
2. proteolytic cleavage
(1) 1ml protein solution adds also original reagent of 10 μ l0.5M, room temperature 30min.
(2) add 27 μ l0.55M alkylating reagents, lucifuge incubated at room 30min.
(3) get 3-10mg albumen, add pancreatin (1:50 pancreatin/albumen), 37 ℃ of digestion are spent the night, and 99 ℃ of heating 5min, end enzymatic activity.The centrifugal 10min of 12000g, abandons precipitation and stays supernatant.
(4) after vacuum decker concentrate drying powdered, then add 1mL deionized water, repeatedly repeat this process 3-4 time.
(5) gained peptide section is got and carried out in right amount liquid chromatography-mass spectrography evaluation.It is the Magic C18AQ of Michrom Bioresources company mutually that liquid phase adopts fixing, 0.1 × 150mm, particle diameter 3 μ m, aperture
μ m; A is 2% acetonitrile mutually, 98% water, and 0.1% formic acid, B is 2% water mutually, 98% acetonitrile, 0.1% formic acid.Adopt 90 minutes gradients, 90 minutes internal linear gradients, from 0 – 35%B, maintain 80%B in 8min, in 12 minutes, maintain 95%B, within 20 minutes subsequently, maintain 0%B.Mass analyzer: LTQ/Orbit rap; Scan pattern: positive ion; Ion gun: the Advance nonoESI of Michrom company spraying; Voltage: 1.6kV; Tube lens voltage: 125V; One-level mass spectrum uses the scanning of total quality number, m/z350-1800, and R=1000,000 (at m/z400), uses m/z=445.120025 as mark in real-time; And ion accumulation to a target value of 106. second order mses, by the scan mode of data dependence, are analyzed ten the highest parent ions of intensity in one-level mass spectrogram, and dynamic mass is got rid of time 27s; Collision induced dissociation energy is set to 35%.
(6) mass spectrogram step (5) being obtained is compared to Africa xenopus and human protein sequence database search engine by Sequest searching algorithm, specifies peptide section sequence, peptide section decorating site and corresponding albumen thereof.Search parameter is: MS1 quality precision ± 10ppm, and MS2 quality precision 1Da, variable modification: Oxidation (M)+15.99492; Peptide section allows 6 variable dynamic embellishment, modifies at most for every kind and occurs 3 times.
Searching storehouse, to obtain result as shown in table 1, and as can be known from Table 1, target protein Rangap-His-SUMO1 is by enriching and purifying.
Table 1. Africa xenopus and human protein sequence database search result
3. antibody enrichment
(1) above-mentioned 2 gained peptide sections are dissolved in to 1-2mL antibody enrichment buffer solution.
(2) add enrichment buffer solution to make final volume reach 1mL the about 200ul antibody of 200 μ g and 100 μ L protein A microballons, the gentle 4h that rotates.
(3) wash three times the centrifugal 30s of 12000g with 1mL enrichment buffer solution.
(4) protein A Beads is joined in peptide section solution e) obtaining to 4 ℃ of gentle 4-8h that rotate.
(5) centrifugal 1000g<10s, carefully removes supernatant.
(6) 1mL PBS washing microballon three times.
(7) remove PBS completely, add 150uL elute soln.
(8) repeated washing 3 times, collects all supernatants, vacuum drying.
4. chemical labeling
(1) sample is dissolved in 50uL 50mM pH7.5 phosphate buffer solution, the final concentration that adds fresh configuration is in 60mM formaldehyde phosphoric acid solution, mixes.
(2) adding final concentration is 10mM sodium borohydride phosphoric acid solution.Mix, 0 ℃ is reacted 2 hours.
(3) Ziptip desalination.Getting appropriate sample is dissolved in containing in 2% acetonitrile solution of 0.1% trifluoroacetic acid, the Ziptip C18 post of crossing with pre-equilibration is adsorbed to state of saturation repeatedly, after the pre-desalination of 2% acetonitrile solution with 0.1% trifluoroacetic acid, with the 50% acetonitrile wash-out repeatedly containing 0.1% trifluoroacetic acid, vacuum drying in sample is managed as for EP.
5. go ubiquitination
(1) sample is dissolved in again to 50mM Tris pH8.0 containing 2mM DTT, 20mM NaCl, in the buffer solution of 100uM SENP1, hybrid reaction.
(2) Ziptip desalination, adds 15uL2% acetonitrile after step is concentrated with 4 (3), and it is to be analyzed that 0.1%FA puts into liquid chromatography automatic sampler.
6. LC-MS analysis
The same 2(5 of conditional parameter of liquid chromatography-mass spectrography).
7. search software analysis and result
The mass spectrogram that step (1) is obtained is compared to human protein sequence database search engine by Sequest searching algorithm, specifies peptide section sequence, peptide section decorating site and corresponding albumen thereof.Search parameter is: MS
1quality precision ± 10ppm, MS
2quality precision 1Da, variable modification: Oxidation (M)+15.99492; Dimethylation (K)+28.0313; N is terminal modified: Dimethylation ,+28.0313; Peptide section allows 6 variable dynamic embellishment, modifies at most for every kind and occurs 4 times.
The peptide section decorating site of having identified is carried out to manual confirmation, obtain the accurate information list that contains SUMO modified protein and decorating site.
List according to above experiment the Africa xenopus Rangap protein related peptide section that Mass Spectrometric Identification goes out as shown in table 2, all peptide section N ends and SENP enzyme are cut front free lysine residue also by chemical modification (amino acid after chemical modification represents to distinguish with unmodified amino acid with lowercase), and lVQMGLLKSEDk, sLLVQMGLLKSEDk, iTSLLVQMGLLKSEDk, vFITSLLVQMGLLKSED, in the peptide sections such as gFQANVFITSLLVQMGLLKSEDk, contain common not by the lysine residue of chemical modification, show that 522 lysine residues of Africa xenopus Rangap albumen are SUMOization decorating site, meet the classical motif feature of SUMOization substrate Ψ KxE, and most of peptide sections are as lVQMGLLk, lTLNHMVQQNYFPk etc., irrelevant with SUMOization site.
Peptide section after IP is cut and directly entered the analysis of liquid chromatography mass combined instrument without chemical modification, SENP enzyme, pass through manual search, find SUMO1 modified peptides section GFQANVFITSLLVQMGLL (ELGMEEEDVIEVYQEQTGG) KSEDK, its second order ms figure as shown in Figure 4, confirms that 522 lysine residues of Africa xenopus Rangap albumen are SUMO1ization decorating site.
Table 2. Africa xenopus Rangap protein related peptide section
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (14)
1. a protein-based ubiquitination site authentication method, is characterized in that, comprises the following steps:
(1) the SUMOization albumen of purifying being carried out to enzyme cuts;
(2) specific antibody is to the enrichment of SUMOization modified peptides section;
(3) chemical labeling is protected the lysine amino residue of non-modifiedization in institute's enrichment peptide section;
(4) go ubiquitination enzymic catalytic reaction to expose adorned lysine on substrate peptide section sequence;
(5) liquid chromatography mass combined instrument is analyzed peptide section sequence;
(6) utilize proteomic image data analysis software coupling to obtain by the peptide section sequence of chemical modification, the lysine sites containing free amine group in its sequence is ubiquitin-like decorating site;
Described SUMOization is modified and is comprised SUMO1, and SUMO2 and SUMO3 ubiquitin-likeization are modified.
2. the protein-based ubiquitination of one according to claim 1 site authentication method, it is characterized in that, the described antibody of step (2) refers to the specific antibody that SUMOization peptide section is modified, through rabbit anteserum immunity gained, specific recognition ubiquitin-like be can distinguish and SUMO1, the corresponding specific amino acid sequence of SUMO2 or SUMO3 modified.
3. the protein-based ubiquitination of one according to claim 1 site authentication method, is characterized in that, the described chemical labeling of step (3) refers to the one in dialkyl group modification, acetyl groupization modification or guanidinated modification.
4. the protein-based ubiquitination of one according to claim 3 site authentication method, is characterized in that, described dialkyl groupization is modified and comprised the steps:
(a) sample is dissolved in pH7.5 buffer solution, adds in the aldehydes solution of fresh configuration, mix;
(b) add the original reagent of going back of fresh configuration, mix, after completion of the reaction with stop bath cancellation reaction;
(c) with the desalination of chromatogram pre-column.
5. the protein-based ubiquitination of one according to claim 4 site authentication method, is characterized in that,
The described aldehydes chemical structural formula of step (a) is R
1cHO, R
1for the C of iodine replacement
1～C
10alkyl, concentration is between 0.01-1M;
The described original reagent of going back of step (b) is the one in sodium borohydride, sodium cyanoborohydride or borine, and concentration is between 0.01-1M; Between temperature of reaction 0-60 ℃; Reaction time 1-48 hour; Described stop bath refers to acid solution, is one or more in hydrochloric acid, formic acid, acetic acid or trifluoroacetic acid solution, and concentration is between 0.1-5%;
The described chromatogram pre-column of step (c) is Ziptip desalting column.
6. the protein-based ubiquitination of one according to claim 3 site authentication method, is characterized in that, described acetylation modification comprises the steps:
(a) sample is dissolved in pH 7.5 buffer solution, adds in the N-acetate substituent solution of fresh configuration, mix;
(b) add the deesterify reaction solution of fresh configuration, mix, with stop bath cancellation reaction;
(c) with the desalination of chromatogram pre-column.
7. the protein-based ubiquitination of one according to claim 6 site authentication method, is characterized in that,
The described N-acetate substituent solution of step (a) is one or more in N-acetate acetyl chloride, N-acetoxyacetic acid, N-acetate succinimide or N-acetate glycocoll, and concentration is between 0.01 – 1M, between temperature of reaction 25-60 ℃; Reaction time 1-24 hour;
The described deesterify reaction solution of step (b) is oxammonium hydrochloride, Hydroxylamine sulfate, N, one or more in N-diethyl hydroxylamine, hydroxylamine nitriate or phosphatic hydroxylamine, concentration is between 0.01 – 1M, described stop bath is one or more in glutamic acid, glycocoll or leucine, and concentration is between 1-100mM;
The described chromatogram pre-column of step (c) is Ziptip desalting column.
8. the protein-based ubiquitination of one according to claim 3 site authentication method, is characterized in that, described guanidinated modification comprises the steps:
(a) by sample dissolution in alkaline buffer solution, add the adjacent Methyl Isourea Sulfate solution of fresh configuration, start chemical reaction;
(b) add acidic buffer solution, adjust pH is to acid cancellation reaction;
(c) with the desalination of chromatogram pre-column.
9. the protein-based ubiquitination of one according to claim 8 site authentication method, is characterized in that,
The described alkaline buffer solution of step (a) is one or more in NaOH, potassium hydroxide, sodium carbonate or ammoniacal liquor, and concentration is between 1-10M; Temperature of reaction between-20-100 ℃, reaction time 1-24 hour;
The described acidic buffer solution of step (b) is one or more in formic acid, acetic acid, butyric acid, acetic acid or trifluoroacetic acid, and concentration is between 0.01%-20%;
The described chromatogram pre-column of step (c) is Ziptip desalting column.
10. the protein-based ubiquitination of one according to claim 1 site authentication method, its feature exists
Go ubiquitination enzymic catalytic reaction to comprise the steps: in described in, step (4)
(a) sample is dissolved in again to ubiquitination enzyme and enzymic catalytic reaction buffer solution, mixes 37 ℃ of reactions;
(b) chromatogram pre-column desalination.
The protein-based ubiquitination of 11. one according to claim 10 site authentication method, is characterized in that,
Described in step (a), go ubiquitination enzyme to refer to SENP, comprise SENP1, one or more in SENP2 or SENP3; Described enzymic catalytic reaction solution is to contain phosphoric acid, Tris-HCl or the Hepes-HCl buffer solution of going back original reagent and sodium chloride, the described original reagent of going back is dithiothreitol (DTT) or beta-mercaptoethanol solution, reduction reagent concentration is between 0.001 – 1M, and sodium chloride concentration is between 0.05 – 1M;
The described chromatogram pre-column of step (b) is Ziptip desalting column.
The application of 12. chemical labelings in identify in protein-based ubiquitination site.
The application of 13. chemical labelings according to claim 12 in identify in protein-based ubiquitination site, is characterized in that, described protein-based ubiquitination site is identified and comprised the steps:
(1) the SUMOization albumen of purifying being carried out to enzyme cuts;
(2) specific antibody is to the enrichment of SUMOization modified peptides section;
(3) chemical labeling is protected the lysine amino residue of non-modifiedization in institute's enrichment peptide section;
(4) go ubiquitination enzymic catalytic reaction to expose adorned lysine on substrate peptide section sequence;
(5) liquid chromatography mass combined instrument is analyzed peptide section sequence;
(6) utilize proteomic image data analysis software coupling to obtain by the peptide section sequence of chemical modification, the lysine sites containing free amine group in its sequence is ubiquitin-like decorating site;
Described SUMOization is modified and is comprised SUMO 1, and SUMO2 and SUMO3 ubiquitin-likeization are modified.
The application of 14. chemical labelings according to claim 12 in identify in protein-based ubiquitination site, is characterized in that, described chemical labeling refers to dialkyl group modification, acetylation modification or guanidinated modification.
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