A proteomic approach for the identification of novel lysine methyltransferase substrates

In the current study, we used the ProtoArray？ platform, containing over 9,500 human proteins, and developed and optimized a system for proteome-wide identification of novel methylation events catalyzed by the protein lysine methyltransferase (PKMT) SETD6. This enzyme had previously been shown to methylate the transcription factor RelA, but it was not known whether SETD6 had other substrates. By using two independent detection approaches, we identified novel candidate substrates for SETD6, and verified that all targets tested in vitro and in cells were genuine substrates.We describe a novel proteome-wide methodology for the identification of new PKMT substrates. This technological advance may lead to a better understanding of the enzymatic activity and substrate specificity of the large number (more than 50) PKMTs present in the human proteome, most of which are uncharacterized.Lysine methylation of proteins plays a key role in many signaling and biological pathways, and disruption of this modification can lead to the development of disease [1,2]. A lysine residue in a given protein can be monomethylated, dimethylated or trimethylated by protein lysine methyltransferases (PKMTs). There are approximately 50 PKMTs known to be present in the human proteome, but the enzymatic activity and substrate specificity of most of them are not known. Despite the importance of lysine methylation in maintaining cellular homeostasis, the development of proteome-wide approaches for detecting this modification has been limited and has proven technically difficult. Most methods aimed at identifying new PKMT substrates use candidate-based or mass-spectrometry approaches [3,4]. Peptide-array technologies are also used to identify new targets and potential consensus sequences for a given PKMT [5,6].In the current study, we used a human protein microarray-based platform (ProtoArray？; Invitrogen Corp., Carlsbad, CA, USA) to identify new substrates for PKMTs. This system contains more than 9,