Abstract
Protein arginine methylation is found in many nucleic acid binding proteins affecting numerous cellular functions. In this study we identified methylarginine-containing proteins in HeLa cell extracts by two-dimensional electrophoresis and immunoblotting with a methylarginine-specific antibody. Protein spots with matched protein stain and blotting signals were analyzed by mass spectrometry. The identities of 12 protein spots as 11 different proteins were suggested. Known methylarginine-containing proteins such as hnRNP A2/B1, hnRNP A1, hnRNP G and FUS were identified, indicating the feasibility of our approach. However, four highly abundant metabolic enzymes that might co-electrophorese with methylarginine-containing proteins were also identified. Other nucleic acid binding proteins hnRNP M, hnRNP I and NonO protein were identified. Recombinant hnRNP M and a peptide with the RGG sequence in hnRNP M could be further methylated in vitro. The immunoblotting results of immunoprecipitated hnRNP I and NonO protein are consistent with arginine methylation in both proteins. In this study we identified methylarginine-containing proteins in HeLa cells through proteomic approaches and the method is fast and robust for further applications.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Antibodies / chemistry
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Antibody Specificity
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Arginine / analogs & derivatives*
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Arginine / analysis
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Arginine / metabolism
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DNA-Binding Proteins
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Electrophoresis, Gel, Two-Dimensional / methods*
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HeLa Cells
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Heterogeneous Nuclear Ribonucleoprotein A1
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Heterogeneous-Nuclear Ribonucleoprotein Group A-B / analysis
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Heterogeneous-Nuclear Ribonucleoprotein Group A-B / metabolism
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Heterogeneous-Nuclear Ribonucleoprotein Group M / analysis
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Heterogeneous-Nuclear Ribonucleoprotein Group M / metabolism
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Humans
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Immunoblotting / methods*
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Methylation
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Molecular Sequence Data
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Nuclear Matrix-Associated Proteins / analysis
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Nuclear Matrix-Associated Proteins / metabolism
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Octamer Transcription Factors / analysis
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Octamer Transcription Factors / metabolism
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Peptide Mapping
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Protein Processing, Post-Translational
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Protein-Arginine N-Methyltransferases / metabolism
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RNA-Binding Proteins / analysis
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RNA-Binding Proteins / metabolism
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Substances
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Antibodies
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DNA-Binding Proteins
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Heterogeneous Nuclear Ribonucleoprotein A1
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Heterogeneous-Nuclear Ribonucleoprotein Group A-B
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Heterogeneous-Nuclear Ribonucleoprotein Group M
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NONO protein, human
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Nuclear Matrix-Associated Proteins
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Octamer Transcription Factors
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RNA-Binding Proteins
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Arginine
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Protein-Arginine N-Methyltransferases