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.
Hung
Chien-Jen
CJ
Department of Biomedical Sciences, Chung Shan Medical University, 110 Sec. 1, Taichung, Taiwan, ROC.
Lee
Yu-Jen
YJ
Chen
Da-Huang
DH
Li
Chuan
C
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