Figure 1: Isomethionine methyl-SILAC (iMethyl-SILAC) strategy.

(a) Methionine is incorporated into proteins and also converted to the methyl group donor S-adenosyl methionine. Isotope-labelled methionines are nearly isobaric, but the distribution of label differs, therefore only methylated peptides will be observed as methyl-SILAC pairs. (b) Isomethionine methyl-SILAC labelling removes ambiguity arising from methionine-containing peptides. Methionine-containing peptides and methylated peptides are observed as indistinguishable methyl-SILAC pairs when cells are labelled by heavy methyl-SILAC (top). When cells are labelled by isomethionine methyl-SILAC, only methylated peptides give rise to methyl-SILAC pairs (bottom). (c) False discovery rate (FDR) among identified arginine-methylated peptides as a function of peptide identification confidence (iProphet probability). Primary T cells were labelled by heavy methyl-SILAC or iMethyl-SILAC. Without corroboration by methyl-SILAC pairs, the FDR rises rapidly to unacceptable levels as the minimum accepted iProphet probability is lowered (red line). By requiring a hit to be corroborated by a methyl-SILAC pair, the FDR is lowered in heavy methyl-SILAC-labelled samples (blue line) and further lowered in iMethyl-SILAC-labelled samples (green line).