The Protein Journal

, Volume 28, Issue 3, pp 139–147

Proteomic Analysis of Methylarginine-Containing Proteins in HeLa Cells by Two-Dimensional Gel Electrophoresis and Immunoblotting with a Methylarginine-Specific Antibody

  • Chien-Jen Hung
  • Yu-Jen Lee
  • Da-Huang Chen
  • Chuan Li
Article

DOI: 10.1007/s10930-009-9174-3

Cite this article as:
Hung, CJ., Lee, YJ., Chen, DH. et al. Protein J (2009) 28: 139. doi:10.1007/s10930-009-9174-3

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.

Keywords

Proteomic analysis Posttranslational modification RNA binding proteins Methylarginine-containing proteins Protein arginine methylation 

Abbreviations

PTM

Posttranslational modification

PRMT

Protein arginine methyltransferase

AdoMet

S-adenosylmethionine

aDMA

Asymmetric dimethylarginine

sDMA

Symmetric dimethylarginine

MMA

Monomethylarginine

2-DE

Two-dimensional electrophoresis

IPG

Immobilized pH gradient

IEF

Isoelectrofocusing

Supplementary material

10930_2009_9174_MOESM1_ESM.doc (46 kb)
Supplementary material 1 (DOC 46 kb)

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Chien-Jen Hung
    • 1
    • 2
  • Yu-Jen Lee
    • 1
  • Da-Huang Chen
    • 1
  • Chuan Li
    • 1
  1. 1.Department of Biomedical SciencesChung Shan Medical UniversityTaichungTaiwan, ROC
  2. 2.Institute of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC