Analytic treatment method for protein methylation and application thereof

A technology for analytical processing and methylation, applied in the field of analysis and processing of protein methylation, can solve problems affecting the specificity of separation, efficient separation of methylated peptides and interference

Inactive Publication Date: 2018-05-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, under the SCX chromatographic separation conditions in the low pH range, unmethylated missed peptides and histidine-containing peptides will also carry some positive charges, which will interfere with the efficient separation of methylated peptides
It is reported that in SCX fractions with relatively concentrated methylated peptides, peptides containing histidine will account for 60-70% of all peptides, which greatly affects the specificity of separation

Method used

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  • Analytic treatment method for protein methylation and application thereof
  • Analytic treatment method for protein methylation and application thereof
  • Analytic treatment method for protein methylation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Investigation of the missing cutting situation of methylation sites: unmodified (EIAQDF-K-TDLR), monomethylated (EIAQDF-K(Me1)-TDLR), dimethylated (EIAQDF-K(Me2)-TDLR ) and trimethylated (EIAQDF-K(Me3)-TDLR) peptides dissolved in 50mM NH 4 HCO 3 (pH 8.0), add trypsin according to the ratio of protein: enzyme (50:1, w / w), incubate at 37°C for 16 hours, and then perform laser-assisted ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. Such as figure 2 As shown, by analyzing the mass spectrograms of different peptides after trypsin digestion, it can be found that the C-terminus of unmodified lysine will be completely cut off, and the C-terminus of monomethylated lysine Only a small proportion of the carbon terminal (C-terminal) will be digested, and the C-terminal of lysine modified by dimethylation and trimethylation cannot be digested by trypsin. This result clearly shows that the carbon terminus (C terminus) of the methylated lysine residue and argini...

Embodiment 2

[0034] The multi-enzyme digestion strategy is used to reduce the proportion of missed cleavage sites: add 10-20mM dithiothreitol (DTT) at a final concentration of 1mg protein sample extracted from HepG2 cells, 1- 3h, then add iodoacetamide (IAA) at a final concentration of 20-40mM, and react in the dark at 20-25°C for 40-60min. The solution was replaced with 50 mM ammonium bicarbonate (pH 8.0) by an ultrafiltration assisted sample processing strategy. Divide 1 mg of the treated protein sample into three parts. The first group of samples was added with trypsin according to the ratio of protein: enzyme (50:1, w / w), and incubated at 37°C for 16 hours; the second group of samples was Add trypsin (trypsin) and lysinase (lys-C) at the ratio of protein: enzyme (50:1, w / w), and incubate at 37°C for 16 hours; / w) was added to trypsin and lysinase (lys-C), and incubated at 37°C for 16 hours. Remove trypsin (trypsin) and lysinase (lys-C) by ultrafiltration, then add 10× activation solu...

Embodiment 3

[0041] Optimization of separation conditions for "SCXtip": Use methylated standard peptides to investigate the optimal separation conditions required for SCXtip separation. 5 methylated standard peptides (EIAQDF-K(Me1)-TDLR; EIAQDF-K(Me2)-TDLR; EIAQDF-K(Me3)-TDLR; SG-R(Me1)-GGNFGFGDSR; N-R(Me2S)-GAGGFGGGGGTR ) was dissolved in 60% (vol / vol) acetonitrile, 40% (vol / vol) 5mM BRUB (pH 2.5) solution, loaded into the SCXtip, and successively with 90% acetonitrile, 5mM BRUB (pH9) solution, 85% Acetonitrile, 5mM BRUB (pH 9) solution, 80% acetonitrile, 5mM BRUB (pH 9) solution, 65% acetonitrile, 5mM BRUB (pH 9) solution, 30% acetonitrile, 5mM BRUB (pH 12) solution bound to SCXtip column Peptides were eluted, and the corresponding elution solutions were collected and analyzed by MALDI-TOF mass spectrometry. image 3 , we can see that in the mass spectrum of the sample loading step, no peptide peaks are seen, indicating that the methylated peptides are all retained on the SCXtip; when d...

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Abstract

The invention relates to an analytic treatment method for protein methylation. By utilization of the characteristic that lysine and arginine modified by methylation can inhibit digestion of trypsin, the invention develops a sample treatment method integrating heavy methyl stable isotope labeling by amino acids in cell culture (hM-SILAC), multi-enzyme digestion and separation of strong cation exchange microcolumn (SCXtip), and applies the sample treatment method to large-scale analysis of methylated proteome. The analytic treatment method has the beneficial effects that by combination with a multi-enzyme digestion strategy, the missed digestion sites of non-methylated sources are effectively reduced; by the separation strategy of the strong cation exchange chromatography with high pH range,the interference of peptide fragments containing histidine is effectively overcome, so that the separation specificity of methylated peptide fragments is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of methylation proteomics in the research direction of proteomics, and specifically relates to an analysis and processing method of protein methylation and its application. Background technique [0002] Protein methylation is catalyzed by S-adenosylmethionine (SAM)-dependent methyltransferases, and is one of the most common post-translational modifications of proteins. Methylation modification plays an important regulatory role in the regulation of many physiological processes. Studies have shown that protein methylation can regulate the intramolecular or intermolecular interactions of target proteins; affect their affinity with RNA, thereby affecting a variety of cellular processes, including transcription regulation, cellular localization, ribosome assembly, and RNA processing , maturation of heterogeneous ribosomal ribosomal proteins (hnRNPs), protein-protein interactions, accuracy of translation, nuclea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34G01N30/96
CPCG01N1/34G01N30/96
Inventor 叶明亮王科云
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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