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Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.

Molecular & cellular proteomics : MCP (2013-10-17)
Ailan Guo, Hongbo Gu, Jing Zhou, Daniel Mulhern, Yi Wang, Kimberly A Lee, Vicky Yang, Mike Aguiar, Jon Kornhauser, Xiaoying Jia, Jianmin Ren, Sean A Beausoleil, Jeffrey C Silva, Vidyasiri Vemulapalli, Mark T Bedford, Michael J Comb
ABSTRACT

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.

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Sigma-Aldrich
SILuRich Anti-IL6 MAb, biotin conjugated, clone IL6-4G1, purified from hybridoma cell culture