CN108445221A - A kind of identification method of protein methylation - Google Patents
A kind of identification method of protein methylation Download PDFInfo
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Abstract
The present invention relates to a kind of new Identification Methods of protein methylation; methylation sites are marked using a kind of novel methionine of mark again; develop one kind and combining methionine cell culture amino acid stable isotope labeling; the peptide fragment analysis method that methylates of the fragmentation spectrogram and the processing of spectral peak demethylation of the peptide fragment that methylates is identified and extracted, and is applied in the scale analysis for the protein group that methylates.The amino acid tag mode introduced in the present invention can distinguish methylate peptide fragment and the peptide fragment comprising methionine, by identifying and extracting the fragmentation spectrogram from the peptide fragment that methylates, and carry out demethylation processing to it, and then obtain the spectrogram of the non-peptide fragment that methylates.Peptide section sequence is determined by database search, and then the spectrogram for the modification peptide fragment that methylates is combined to be determined decorating site.This method need not preset modified types, thus known all types that methylate can be carried out while be analyzed.
Description
Technical field
It methylates proteomic techniques field the invention belongs to proteomics research direction, and in particular to methylate egg
The new Identification Method of white matter group.
Background technology
The modification that methylates of protein is relied on by S-adenosylmethionine (S-adenosyl methionine, SAM)
What methyl transferase catalytic generated, be one of most common protein post-translational modification.Modification methylate in many physiology mistakes
Important regulating and controlling effect is played in the regulation and control of journey.Research shows that:Albumen, which methylates, can adjust target protein intramolecular or divide
Interaction between son;The affinity interaction for influencing they and RNA, to affect various kinds of cell process, including it is transcriptional regulatory, thin
Born of the same parents' positioning, ribosomes assembly, RNA processing, the maturation of heterogeneous RNA ribosomal protein (hnRNPs), protein-protein phase
Interaction, the accuracy of translation, nucleus transport, protein nucleic acid transport and metabolism and Cellular Signaling Transduction Mediated etc..Due to first
The diversity and complexity of baseization modification identify that methylation sites are still very difficult, this master in proteomics level
If because the modification that methylates of protein can be happened on up to 8 kinds of amino acid residues, 11 kinds of methylation patterns are contained.
The processing strategy of traditional posttranslational modification is that posttranslational modification type to be studied is set as variable modification.Thus carrying out
Methylate decorating site identification when, if these modified types that methylate are both configured to variable modification, to be arranged simultaneously very
More variable modifications, searching library space can be very big, and false positive rate also can be very high.
Methionine is the essential amino acid that mammalian cell itself cannot synthesize, and synthesis S-adenosylmethionine
Precursor.If the methionine of isotope labelling is marked in addition in cell culture again, the methyl for marking isotope labelling again can be
It is transferred on the side chain of target amino acid with the help of transmethylase.If marked with the methionine of different isotopics
Remember that the methylation sites on albumen, the light mark form of the same peptide fragment that methylates and the parent ion molecular weight of mark form again can exist admittedly
Fixed difference, and this molecular weight difference is to be determined by the number of methyl modification on peptide fragment, thus can pass through this molecule
Amount difference is come the number for the modification that judges to methylate on peptide fragment.But (the document 1 in traditional labeling method:S.-E.Ong,
G.Mittler,M.Mann,Identifying and quantifying in vivo methylation sites by
1 (2004) 119-126 of heavy methyl SILAC, Nat Methods), for the peptide fragment containing methionine and contain first
The peptide fragment of baseization modification, the molecular weight difference that isotope labelling introduces is identical, thus cannot carry out area to this two classes peptide fragment
Point.The present invention combining methionine cell culture amino acid stable isotope labeling using one kind for the first time, identifies the peptide that methylates
The analysis method of the fragmentation spectrogram and spectral peak demethylation of section, and it is applied to the scale analysis for the protein group that methylates
In.By identifying and extracting the fragmentation spectral peak of peptide fragment of methylating, and demethylation processing is carried out to it, and then obtains non-methylate
The spectral peak of peptide fragment greatly simplifies database search process.And then combine the spectrogram for the modification peptide fragment that methylates can be to modification
Site is positioned.This method need not preset modified types, thus a variety of modifications that methylate can be carried out while be analyzed.
Invention content
The purpose of the present invention is to provide a kind of collection methionine cell culture amino acid stable isotope marks of high throughput
Note technology identifies that the fragmentation spectrogram for the peptide fragment that methylates and spectral peak demethylation are newly square in the Proteomic analysis that methylates of one
Method.
The analyzing novel methods of the protein group sample proposed by the present invention that methylates are utilized light mark and methylate and peptide fragment and mark again
The molecular weight difference to methylate between peptide fragment is directly linked this characteristic with the number with methyl, realizes based on the peptide that methylates
The identification and the processing of spectral peak demethylation of section spectrogram.
It is as follows:
(1) cell is cultivated containing light mark methionine and again in mark methionine DMEM culture solutions respectively;
(2) cell for obtaining above-mentioned steps (1) ultrasonic wave added in lysate is broken, according to protein content 1:1 ratio
Mixing;
(3) protein sample obtained in above-mentioned steps (2) is subjected to digestion processing;
(4) protein enzymatic hydrolyzate obtained in above-mentioned steps (3) is subjected to high resolution mass spectrum analysis.
(5) the fragmentation spectrogram of the peptide fragment that methylates in the mass spectrometric data obtained to above-mentioned steps (4) is identified, and carries out
The processing of spectral peak demethylation, and then database search is carried out to determine peptide section sequence.
(6) spectrogram before the peptide section sequence and demethylation that are obtained in step (5) is combined to determine the modification that methylates
Site;
Cell used in the present invention be selected from human archeocyte, such as HEK293 cells, HepG2 cells, HeLa cells or
Jurkat cell.
Cell culture system described in step (1) is the l-methionine containing 0.2mM or l-methionine-carboxylic
Base-13C, methyl D3DMEM culture mediums, and be added 10% dialysis treatment fetal calf serum.
Cell cracking system described in step (2) be containing 6-8M urea, 1-2%v/v Triton X-100s,
60-70mM dithiothreitol (DTT)s, 1-2mM phenylmethylsulfonyl fluorides, 1-2%v/v protease inhibitors, 50-100mM Tris-Cl (pH
7.5) buffer solution..
Final concentration of 10-20mM dithiothreitol (DTT)s, 37-60 is added in the protein example obtained to 1mg above-mentioned steps (2)
Then final concentration of 20-40mM iodo-acetamides are added in 1-3h in DEG C water-bath, 20-25 DEG C is protected from light 40-60min, is added 4
The buffer solution of the 50mM Tris-Cl (pH 8.1) of times volume, according to albumen:Enzyme mass ratio is 50:1 is added trypsase
(trypsin), it is digested 16 hours under the conditions of 37 DEG C.Gained peptide fragment is lyophilized at room temperature, the protein group sample that obtains that treated.
The peptide fragment obtained in above-mentioned steps (3) is redissolved and carries out LC-MS/MS points of RP in the formic acid of 0.1% volumetric concentration
Analysis.
The fragmentation spectrogram of the peptide fragment that methylates in the mass spectrometric data that is obtained in above-mentioned steps (4) is identified, and is carried out
The processing of spectral peak demethylation, and then database search is carried out to determine peptide section sequence.
The decorating site that methylates is determined in conjunction with the spectrogram before the peptide section sequence and demethylation obtained in step (5);
The processing method is applied to the proteomics efficient analysis that methylates, the corresponding modification peptide fragment that methylates can be obtained
Sequence information, which can identify in conjunction with subsequent site determine that the type for the modification that methylates, this method can be used for turning over
Proteome analysis is modified after translating.
Description of the drawings
Below in conjunction with the accompanying drawings and embodiment the present invention is described in further detail:
Fig. 1 is the flow chart of the new Identification Method of protein methylation.It is utilized respectively light mark methionine and marks first sulphur again
Propylhomoserin marks methylation sites, and smudge cells extract albumen, according to protein content 1:1 ratio mixing.Then digestion is carried out
Processing, and carry out subsequent mass spectral analysis;Fragmentation spectrogram for the peptide fragment that methylates occurred in pairs, point that each methyl introduces
Son amount difference is 3Da;Fragmentation spectrogram for the peptide fragment with methionine occurred in pairs, point that each methionine introduces
Son amount difference is 4Da.Therefore this two classes peptide fragment can be distinguished.
Fig. 2 is the demethylation processing procedure of spectral peak.Sentence according to gently marking spectral peak and marking the molecular weight difference between spectral peak again
The number of the disconnected modification group that methylates for including, and derive the molecular weight of the spectral peak of corresponding non-modified peptide fragment.
Methylate the non-modified peptide fragment for modifying non-modified peptide fragment fragmentation spectrogram and synthesis that spectrogram is derived according to Fig. 3
Fragmentation spectrogram searches library result.The matching result of the two spectrograms is very close, it was demonstrated that having for spectral peak demethylation strategy
Effect property.
Specific implementation mode
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.
Embodiment 1
Scale analysis of the spectral peak demethylation strategy for the proteomics that methylates:By HEK293 cells respectively containing
Cultivated for eight generations (at least in the DMEM culture mediums of the fetal calf serum of the dialysis treatment for being added to 10% of the l-methionine of 0.2mM
Eight generations), in l-methionine-carboxyl-containing 0.2mM13C, methyl D3The dialysis treatment that is added to 10% fetal calf serum
It was cultivated in DMEM culture mediums for eight generations (at least eight generations);Cell is collected respectively and respectively in lysate (8M urea, the poly- second of 1%v/v two
Alcohol octyl phenyl ether, 65mM dithiothreitol (DTT)s, 1mM phenylmethylsulfonyl fluorides, 1%v/v protease inhibitors, 50mM Tris-Cl
The buffer solution of pH 7.5) in ultrasonic wave added it is broken;According to protein content 1:1 ratio mixes light standard specimen product and weight standard specimen product.
Final concentration of 20mM dithiothreitol (DTT)s are added into 2mg protein mixing samples, then 2h in 37 DEG C of water-baths is added final concentration of
40mM iodo-acetamides, 25 DEG C are protected from light 40min, and the buffer solution of the 50mM Tris-Cl (pH 8.1) of 4 times of volumes is added,
According to albumen:Enzyme mass ratio is 50:1 is added trypsase (trypsin), is digested 16 hours under the conditions of 37 DEG C, by gained peptide
Section sample desalination is simultaneously lyophilized at room temperature.Linear salt gradient (starting using strong cation exchange chromatography by sample at 36 minutes
Salinity is 0mM KCl;Termination salinity is 250mM KCl) in be divided into 36 fractions (fraction per minute), desalination and
It is lyophilized at room temperature, obtained peptide fragment is redissolved and carries out RPLC-MS/MS analyses in the formic acid of 0.1% volumetric concentration.Pass through identification
The spectrogram of the peptide fragment that methylates in mass spectrum file simultaneously carries out spectral peak demethylation processing, user's database (under be downloaded from
Uniprot, http://www.uniprot.org/) spectrogram search is carried out to determine the amino acid sequence of modification peptide fragment, Jin Erjie
The spectrogram of modification peptide fragment is closed to determine decorating site, we have successfully identified 512 first on 352 methylation sites
Base is as a result, cover 10 kinds of types that methylate in addition to cysteine methylates, it was demonstrated that this analysis method has very
Strong applicability.The present invention is a kind of new Identification Method of protein methylation, and having developed one kind, to combine methionine thin
Born of the same parents cultivate amino acid stable isotope labeling, identify and extract the peptide fragment that methylates fragmentation spectrogram and spectral peak demethylation processing
Methylate peptide fragment analysis method, and is applied in the scale analysis for the protein group that methylates.This method need not be pre-
If modified types, thus a variety of methylate can be carried out while be analyzed.This identification method that methylates is the protein that methylates
The research and development that group is learned, provides a new methods and techniques platform.
Claims (10)
1. a kind of identification method of protein methylation, it is characterised in that:
(1) utilize again mark methionine cell culture amino acid stable isotope labeling technology by the modification that methylates in protein
Group marks again;It will be in protein using light mark methionine cell culture amino acid stable isotope labeling technology
The modification group that methylates carries out light mark label;
(2) cell for obtaining step (1) is according to cell number 1:Albumen is extracted in 1 ratio mixing, or in extraction albumen
Afterwards, according to protein content 1:1 ratio mixing;
(3) trypsase Trypsin enzymolysis is carried out to the protein sample that step (2) obtains and carries out high resolution mass spectrum analysis;
(4) the fragmentation spectrogram of the peptide fragment that methylates in the mass spectrum file obtained to step (3) is identified, and carries out spectral peak to it
Demethylation handles to obtain the spectrogram of corresponding non-modified peptide fragment;
(5) database search is carried out to determine the sequence of peptide fragment to the spectrogram that step (4) obtains;
(6) spectrogram before the peptide section sequence and demethylation that are obtained in step (5) is combined to determine the decorating site that methylates.
2. identification method according to claim 1, it is characterised in that:
Step (1) (2) concrete operations are that cell is being marked methionine or again the DMEM trainings of mark methionine containing light respectively
It is cultivated in nutrient solution;The cell of two kinds of obtained flag states is broken respectively at ultrasonic wave added in lysate, and according to protein content 1:1
Mass ratio mixing, or the obtained cell of two kinds of flag states is according to cell number 1:1 ratio is mixed in lysate
Ultrasonic wave added is broken.
3. identification method according to claim 1 or 2, it is characterised in that:
The system of the cell culture is the l-methionine containing 0.1-0.3mM or l-methionine-carboxyl-13C, methyl-
D3DMEM culture mediums, and be added the dialysis treatment molecule of the following molecular weight of 10K Da (removal) of final volume concentration 5-20%
Fetal calf serum;
The system of the cell pyrolysis liquid is containing 6-8M urea, 1-2%v/v Triton X-100s, bis- sulphur of 60-70mM
Threitol, 1-2mM phenylmethylsulfonyl fluorides, 1-2%v/v protease inhibitors, the buffering of 50-100mM Tris-Cl (pH 7.5)
Solution.
4. identification method according to claim 1, it is characterised in that:
The operating procedure of enzymolysis processing is in step (3):Bis- sulphur threoses of final concentration of 10-20mM are added into protein example
Then final concentration of 20-40mM iodo-acetamides are added in alcohol, 1-3h in 37-60 DEG C of water-bath, 20-25 DEG C is protected from light 40-
The buffer solution of the 25-100mMTris-Cl (pH 7.5-8.5) of 4-6 times of volume is added, according to albumen in 60min:Enzyme mass ratio
It is 50:1‐100:1 is added trypsase (trypsin), is digested 16-20 hours under the conditions of 35~38 DEG C.
5. identification method according to claim 1, it is characterised in that:Step (3) redissolves the obtained peptide fragment sample that methylates
RP LC-MS/MS analyses are carried out in formic acid.
6. identification method according to claim 1, it is characterised in that:
When identification methylates the spectrogram of peptide fragment, for the spectrogram in the peptide fragment source that methylates occurred in pairs, each methyl
The molecular weight difference that group introduces is 2.9-3.1Da;Fragmentation spectrogram for the peptide fragment comprising methionine occurred in pairs, often
The molecular weight difference that a methionine introduces is 3.9-4.1Da.
7. identification method according to claim 1, it is characterised in that:
The parent ion molecular weight of demethylation peptide fragment is that the peptide fragment that methylates subtracts the molecular weight after methyl group.
8. identification method according to claim 1, it is characterised in that:
In the spectrogram that methylates of successful matching, two level spectral peak can be divided into two classes, and one kind is the identical spectral peak of molecular weight, this kind of
Spectral peak is the fragment ion not comprising the decorating site that methylates, and in the spectral peak of demethylation, the molecular weight of this kind of spectral peak is protected
It holds constant;The another kind of spectral peak for fixed member amount difference, this kind of spectral peak are the fragment ion for including the decorating site that methylates,
In the spectral peak of demethylation, the molecular weight of this kind of spectral peak is the molecular weight after subtracting methyl group.
9. identification method according to claim 1, it is characterised in that:
It is light to mark spectrogram and the again difference and reason of the parent ion quality of mark spectrogram when identification methylates the fragmentation spectrogram of peptide fragment
Tolerance by the deviation of difference value is that 5-10p.p.m. gently marks peptide fragment and the again tolerance of the deviation of the retention time of mark peptide fragment
Range is ± 100 seconds;The tolerance of the deviation of the light parent ion signal strength marked peptide fragment and mark peptide fragment again is within twice.
10. identification method according to claim 1, it is characterised in that:
The cell is HEK293 cells.
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Cited By (3)
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CN111208244A (en) * | 2018-11-22 | 2020-05-29 | 中国科学院大连化学物理研究所 | Antibody-independent protein methylation modification enrichment analysis method |
CN112852914A (en) * | 2019-11-28 | 2021-05-28 | 中国科学院大连化学物理研究所 | Method for weakening drug resistance of tumor cells |
CN114994160A (en) * | 2022-05-24 | 2022-09-02 | 天津医科大学 | Analysis method for detecting formaldehyde-treated protein/polypeptide/amino acid food/product |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN111208244A (en) * | 2018-11-22 | 2020-05-29 | 中国科学院大连化学物理研究所 | Antibody-independent protein methylation modification enrichment analysis method |
CN112852914A (en) * | 2019-11-28 | 2021-05-28 | 中国科学院大连化学物理研究所 | Method for weakening drug resistance of tumor cells |
CN112852914B (en) * | 2019-11-28 | 2023-01-13 | 中国科学院大连化学物理研究所 | Method for weakening drug resistance of tumor cells |
CN114994160A (en) * | 2022-05-24 | 2022-09-02 | 天津医科大学 | Analysis method for detecting formaldehyde-treated protein/polypeptide/amino acid food/product |
CN114994160B (en) * | 2022-05-24 | 2023-09-15 | 天津医科大学 | Analysis method for detecting formaldehyde-treated protein/polypeptide/amino acid food/product |
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