The methylation of proteins is of increasing biological interest. It is predominantly found on lysine and arginine residues, but has also been found on histidine, glutamic acid and on the carboxyl groups of proteins (reviewed in Grillo and Colombatto 2005) . Methylation of lysine involves the addition of one to three methyl groups on the amino acid's ε-amine group, to form mono-, di- or tri-methyllysine. Its function is best understood in histones. Methylation on the tails of histone proteins, in conjunction with acetylation and phosphorylation, controls their interaction with other proteins, affects chromatin compaction and the up- or down-regulation of gene expression . For S. cerevisiae, lysine methylation is found in histone H3 and histone H4 . Tri-methylation at H3K4 and H3K36 is positively correlated with gene activity , while H3K79 are involved in gene silencing [5, 6]. Histone H3K79 methylation is evolutionarily conserved and is involved in several pathways, including Sir protein-mediated heterochromatic gene silencing . meiotic checkpoint control  and in the G1 and S phase DNA damage checkpoint functions of Rad9p [9, 10]. While studies of lysine methylation have mainly focused on histone proteins, several non-histone proteins are also known to be lysine-methylated. They are mainly ribosomal proteins or proteins involved in protein translation , and include Rpl12p [12, 13], Rpl23p [12, 14], Rpl42p , and eEF1Ap .
The methylation of arginine involves the addition of one or two methyl groups to the amino acid's guanidino group, forming mono- or di-methylarginine. It is predominantly known to be associated with RNA regulation and processing . In S. cerevisiae, Hmt1p is a type 1 arginine methyltransferase that catalyses the formation of mono- and asymmetric di-methylarginine. This enzyme is known to methylate a number of proteins that contain an RGG-motif; these include Npl3p, Hrp1p, Nab2p, Gar1p, Nop1p, Nsr1p, Yra1p, Sbp1p, and Hrb1p. These proteins have been implicated in poly(A)+ mRNA binding, processing and export , ribosome biogenesis [18–20] and gene silencing . Moreover, methylation is required for the nuclear export of RNA binding proteins Npl3p, Hrp1p, and Nab2p [22, 23]. The repeated RGG-motif was known as a RNA-binding motif , and this also supports the role of arginine methylation in the regulation of mRNA binding . The methylation of nuclear shuttling proteins is suggested to weaken their binding with cargo proteins and disrupt their export from the nucleus . Arginine methylation is also known to facilitate or block protein-protein interactions. Arginine methylation of SmB protein facilitates the binding of tudor domains in SMN, SPF30, and TDRD3 proteins . In contrast, arginine methylation of Sam68 blocks the interaction of nearby proline-rich motif with an SH3 domain, but not to a WW domain . More examples on methylarginine-regulated interactions are reviewed in McBride and Silver (2001)  and Bedford and Clarke (2009) .
There have been several studies to identify arginine or lysine-methylated proteins on a proteome-wide scale. In the first of these studies, arginine-methylated protein complexes were purified from HeLa cell extracts using anti-methylarginine antibodies specific against RG-rich sequences . This resulted in the identification of over 200 arginine-methylated proteins, involved in pre-mRNA processing, protein translation, and DNA transcription. However the actual methylation sites on these proteins remain unknown . The second study utilised stable isotope labelling by amino acid in cell culture (SILAC), in which [13CD3]methionine was converted to [13CD3]S-adenosyl methionine, the substrate for arginine and lysine methylation . Advantages of this method included increased confidence of identification, a capacity to distinguish between trimethylation and acetylation which are near-isobaric, and the ability to quantify the relative changes in methylation status of a protein between two samples. In combination with anti-methyllysine and anti-methylarginine antibody immunoprecipitation techniques, Ong et al. (2004)  was able to identify methylation on histones from HeLa cell extracts, such as on histone H3K27. Around 30 other proteins were also found to be methylated at RG-rich motifs and most of these proteins are RNA binding or associated with mRNA processing pathways. The third study used anti-methyllysine antibodies to search for organ-specific lysine methylation in Mus musculus . Proteomic analysis of brain tissue extract by 2-D PAGE, western blotting, and MALDI-ToF peptide mass fingerprinting identified the following lysine-methylated proteins: neurofilament triplet-I protein, Hsc70 protein, creatine kinase, α-tubulin, α-actin, β-actin, and γ-actin. Furthermore, α-actin and creatine kinase were found to be methylated in muscle tissue.
The use of tandem mass spectrometry to discover new protein post-translational modifications is common . However, peptide mass fingerprinting can also be used to search for new PTM sites . The FindMod program  caters for this approach. It requires peptide mass spectra from a mostly pure protein, for example a spot from 2-D gel, and examines experimental peptide masses for differences in mass with theoretical peptides for that protein that correspond to post-translational modification. Peptides that are potentially modified are checked to see if they contain amino acids that can carry the modification. Where very high accuracy peptide mass measurements can be made, for example with new instruments like the prOTOF2000, high confidence predictions are possible. Parent-ion masses from tandem mass spectrometry data can also be used in FindMod, where it may serve as an initial screen for PTMs before employing more sophisticated and computationally expensive methods [36, 37].
Here we describe a strategy for the discovery of methylation on a global scale, using peptide mass fingerprinting data, and implement this to search for methylated lysine and arginine residues in the yeast proteome. A proteome-scale set of MALDI-ToF mass spectra  was analysed for putative methylated peptides. The application of 5 filters yielded high-confidence methylation sites that were then further investigated to understand where they are found in protein sequences and their likely function.