Proteomic analysis of methylarginine-containing proteins in HeLa cells by two-dimensional gel electrophoresis and immunoblotting with a methylarginine-specific antibody.
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.
Department of Biomedical Sciences, Chung Shan Medical University, 110 Sec. 1, Taichung, Taiwan, ROC.
Chien-Jen
CJ
Hung
Yu-Jen
YJ
Lee
Da-Huang
DH
Chen
Chuan
C
Li
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000918:Antibody Specificity; D001120:Arginine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D004268:DNA-Binding Proteins; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000032:analysis / Q000378:metabolism; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M / Q000032:analysis / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D034521:Nuclear Matrix-Associated Proteins / Q000032:analysis / Q000378:metabolism; D050810:Octamer Transcription Factors / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D004268:DNA-Binding Proteins; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D016601:RNA-Binding Proteins; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases