BACKGROUND
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   <cit> .
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  <cit> .
for s. cerevisiae, lysine methylation is found in histone h <dig> and histone h <dig>  <cit> .
tri-methylation at h3k <dig> and h3k <dig> is positively correlated with gene activity  <cit> , while h3k <dig> are involved in gene silencing  <cit> .
histone h3k <dig> methylation is evolutionarily conserved and is involved in several pathways, including sir protein-mediated heterochromatic gene silencing  <cit> .
meiotic checkpoint control  <cit>  and in the g <dig> and s phase dna damage checkpoint functions of rad9p  <cit> .
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  <cit> , and include rpl12p  <cit> , rpl23p  <cit> , rpl42p  <cit> , and eef1ap  <cit> .
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  <cit> .
in s. cerevisiae, hmt1p is a type  <dig> 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+ mrna binding, processing and export  <cit> , ribosome biogenesis  <cit>  and gene silencing  <cit> .
moreover, methylation is required for the nuclear export of rna binding proteins npl3p, hrp1p, and nab2p  <cit> .
the repeated rgg-motif was known as a rna-binding motif  <cit> , and this also supports the role of arginine methylation in the regulation of mrna binding  <cit> .
the methylation of nuclear shuttling proteins is suggested to weaken their binding with cargo proteins and disrupt their export from the nucleus  <cit> .
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, spf <dig>  and tdrd <dig> proteins  <cit> .
in contrast, arginine methylation of sam <dig> blocks the interaction of nearby proline-rich motif with an sh <dig> domain, but not to a ww domain  <cit> .
more examples on methylarginine-regulated interactions are reviewed in mcbride and silver   <cit>  and bedford and clarke   <cit> .
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  <cit> .
this resulted in the identification of over  <dig> arginine-methylated proteins, involved in pre-mrna processing, protein translation, and dna transcription.
however the actual methylation sites on these proteins remain unknown  <cit> .
the second study utilised stable isotope labelling by amino acid in cell culture , in which methionine was converted to s-adenosyl methionine, the substrate for arginine and lysine methylation  <cit> .
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.
<cit>  was able to identify methylation on histones from hela cell extracts, such as on histone h3k <dig>  around  <dig> 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  <cit> .
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, hsc <dig> 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  <cit> .
however, peptide mass fingerprinting can also be used to search for new ptm sites  <cit> .
the findmod program  <cit>  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 protof <dig>  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  <cit> .
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  <cit>  was analysed for putative methylated peptides.
the application of  <dig> filters yielded high-confidence methylation sites that were then further investigated to understand where they are found in protein sequences and their likely function.
RESULTS
large-scale methylation discovery in yeast peptide mass spectra
findmod was used to analyse peptide mass spectra for  <dig>  yeast proteins out of a total ~ <dig>   for the presence of mono- and di-methylation.
a tailor-made mass tolerance was calculated for each spectrum to reduce spurious peptide matches; the average of this for all spectra was ±  <dig>  da.
of all the  <dig>  findmod queries, there were  <dig>  matches to potentially methylated peptides .
five filtering strategies, used sequentially, were then applied to this set to find methylation sites of very high confidence.
the first filter removed peptides that matched to unmodified peptide sequences as these peptide masses are likely to be unmodified peptides.
conversely, peptides masses that did not match to unmodified peptide sequences are likely to be modified, and these were analysed with the second filter.
the second filter removed any peptides that contained d or e residues, as artifactual methylation may result from partial methyl esterification of d or e residues  <cit> .
the third filter was designed to take advantage of redundancy within each findmod output, by removing one-off or spurious mass spectra.
it searched for modifications that were found in two or more overlapping peptides , and took advantage of the reduced efficiency of tryptic cleavage at methylated residues  <cit> , where overlapping peptides with missed cleavages were likely to be found.
the fourth filter reduced findmod false positives by considering whether modifications found by findmod were unambiguous or ambiguous.
an unambiguous modification had only one findmod match against one query peptide mass , an ambiguous modification had more than one match against a query mass .
for the peptide to be included in the final set of methylated peptides, at least one peptide in the overlapping peptides had to be an unambiguous peptide match.
the use of these  <dig> filters resulted in  <dig> high confidence methylated peptides, from  <dig>  initial low confidence matches .
while overlapping peptides helped localise methylation sites to one or more peptides, they did not necessarily localise the methylation to one amino acid.
to address this, we used a fifth filter.
when two or more modified peptides that passed filters 1- <dig> were also found to overlap and share the same modification site, the modification was classified as high confidence and kept.
note that any results for lysine trimethylation were discarded from the study since it is near-isobaric to lysine acetylation.
from this filtering process, we found  <dig> lysine-methylated proteins with  <dig> lysine methylation sites:  <dig> with mono- and  <dig> with di-methylation.
similarly, we found  <dig> arginine-methylated proteins with  <dig> arginine methylation sites:  <dig> with mono- and  <dig> with di-methylation.
there were  <dig> proteins that contained both arginine and lysine methylation.
the list of high confidence methylated proteins and methylation sites are shown in table  <dig>  additional information on these high confidence methylated peptides and methylation sites are shown in additional file  <dig> and additional file  <dig> correspondingly.
a: m, monomethylated; d, dimethylated
confirmation of findmod protein methylation
to establish the accuracy of our methylation discovery approach, we theoretically digested all known methylated proteins in swiss-prot and analysed the resulting peptides with our findmod approach.
we supplemented this with a larger set of theoretically methylated proteins.
the average true positive rate for findmod at  <dig>  da was 89%.
for methylation sites in swiss-prot, findmod had a true positive rate of 100% for monomethyl-k, 98% for dimethyl-k, and 76% for dimethyl-r .
the true positive rate for monomethyl-r could not be accurately estimated since the number of test cases was insufficient for accurate evaluation.
similarly, the true positive rate for the artificial methylation set was 78% for monomethyl-k, 89% for dimethyl-k, and 90% for both monomethyl-r and dimethyl-r .
additional results for the evaluation of the true positive rate of findmod are shown in additional file  <dig>
a: n.d. - not determined
to further assess the accuracy of the findmod approach, methylation sites discovered by findmod were cross-referenced with known methylation sites in the literature and databases.
whilst only a small number of proteins are documented as methylated in the literature, we confirmed  <dig> proteins  as methylated .
if we included methyl-lysine sites in peptides containing d and e, we also confirmed the methylation of tef1p and rpl23p.
this included  <dig> lysine methylation sites  from tef1p, and  <dig> lysine dimethylation site  from rpl23p  <cit>  .
furthermore, we found  <dig> methylated ribosomal proteins in s. cerevisiae, consistent with the presence of methylation sites in ribosomal proteins of eukaryotes, such as s. cerevisiae  <cit> , s. pombe  <cit> , a. thaliana  <cit> , and human  <cit> .
a: proteins previously known to be methylated in yeast and in other organisms
discovery rate of methylated peptides, unmodified peptides, and lysine and arginine-methylated residues
the discovery rate of a peptide is the frequency of protein identifications in which a particular peptide is observed.
methylated peptides with low discovery rates are likely to be sub-stoichiometric and partially methylated.
it was predicted that there should be many more unmodified peptides than methylated peptides, and that methylated peptides will have a lower discovery rate since they are likely to be sub-stoichiometric.
the discovery rate of high confidence methylated peptides was found to be significantly lower than that of unmodified peptides .
the median discovery rate for unmodified peptides was  <dig> , and the median value for arginine and lysine methylated peptides was  <dig> .
to check that the lower discovery rate of methylated residues was not due to differences in peptide ionisation efficiency, we examined if there was a correlation between the discovery rates of methylated and unmodified residues.
in the set of results, there were  <dig> methylated residues for which the corresponding unmodified residues were also seen.
the discovery rate of methylated residues was significantly but weakly correlated with the discovery rate of matching unmodified residues , consistent with expected.
a list of methylated proteins and the methylation sites discovered by findmod is shown in table  <dig>  the discovery rate of all high confidence methylated peptides and methylation sites are shown in additional file  <dig> and additional file  <dig> correspondingly.
biological function, sub-cellular localization, abundance and half-life of methylated proteins
methylated proteins are known to be involved in several pathways, such as translation  <cit>  and rna processing  <cit> .
to investigate the function of the methylated proteins from yeast, gene ontology  annotations for all yeast methylated proteins from findmod analysis and swiss-prot were compared to non-methylated yeast proteins .
it was found that a number of biological processes were enriched with very high statistical significance, specifically translation, ribosome biogenesis and assembly, rna metabolic process, and organelle organization and biogenesis.
the molecular function of structural activity, rna binding, and translation regulator activity were also significantly enriched.
as may be expected from the above, methylated proteins were significantly enriched in the cellular components of the ribosome and cytoplasm.
a: number of methylated proteins with this go slim term
b: number of non-methylated proteins with this go term
c: number of methylated proteins with other go slim term
d: number of non-methylated proteins with other go slim term
protein abundance data from ghaemmaghami et al.
<cit>  was used to compare the abundance of methylated proteins to non-methylated proteins.
this was used to determine if lower abundance proteins, more likely to be involved in signal transduction and regulation  <cit> , are methylated.
methylated proteins were found to have a higher median abundance of  <dig>  as compared to non-methylated proteins, which had a median abundance of  <dig> .
despite this, several methylated proteins of low abundance were seen including  <dig> proteins of less than  <dig> molecules per cell.
these included snf2p , snu114p , mrpl20p , and rpl3p .
examples of proteins with high abundance are rp1bp  and tdh3p .
the methylation of lysine residues has been suggested to block their ubiquitination, leading to a longer protein half-life  <cit> .
to investigate this possibility, protein half-life data from belle et al.
<cit>  was used to compare the half-life of lysine-methylated proteins to non-methylated proteins.
interestingly, we found methylated proteins had a longer median half-life of  <dig> minutes, as compared to  <dig> minutes for non-methylated proteins .
a striking difference between the methylated and non-methylated proteins was the absence of a group of proteins with very short half-life .
despite this, our approach also identified  <dig> methylated proteins with half-life less than  <dig> minutes.
examples of methylated proteins with shorter half-lives are rrp5p , ski3p , and snu114p .
examples of proteins with long half-life are utp22p  and atp2p , although we note that these numbers may be erroneous estimations in the belle et al.
study   <cit> .
although the abundance and half-lives of methylated proteins could be analysed more precisely by comparing methylated proteins to other proteins from the same go slim biological process, this approach was limited by the relatively small number of methylated proteins  in the dataset.
methylated proteins mapped to  <dig> gene ontology biological process categories, with an average of  <dig> proteins per category, which was unsuitable for appropriate statistical analyses.
interplay of methylation and other post-translational modifications
to see if lysine methylation might block ubiquitination, the ubipred software  <cit>  was used to predict if known methylated lysine sites are also subject to ubiquitination.
the ubipred software has an accuracy of  <dig> % and is thus sufficiently reliable for this test.
it was found that 43% of high-confidence lysine methylation sites were also predicted to be ubiquitination sites.
this result lends support to the hypothesis that methylation might block ubiquitination, potentially prolonging the half-life of lysine-methylated proteins.
it has recently been reported that the methylation of arginine can regulate the phosphorylation  of some proteins  <cit> .
to investigate whether there is evidence of interplay between arginine methylation and phosphorylation in s. cerevisiae, we examined the proportion of arginine-methylated proteins that are known to be phosphorylated in databases and in the literature.
it was found that 94%  of arginine-methylated yeast proteins are known to be phosphorylated.
this is a considerable increase over the 38%  of all s. cerevisiae proteins known to be phosphorylated and suggests a possible interplay of arginine methylation and phosphorylation  <cit> .
arginine and lysine methylation motifs
to determine if methylation sites are enriched in specific sequence-motifs, all yeast methylation sites from findmod analysis and the swiss-prot database were analysed to find enriched sequence-motifs.
methionine was found to be at position - <dig> from lysine methylation in  <dig> findmod sites and two additional methylation sites previously documented in s. cerevisiae .
this presence of methylation was of very high statistical significance  as compared to that expected in any random sequence of yeast proteins.
by contrast, residues found to be significantly enriched adjacent to arginine methylation included w at position - <dig> , and g at position - <dig> .
while it was previously known that arginine methylation is found in rgg motifs, wooderchak et al.
<cit>  showed that arginine methylation is also found in rxg and rgx motifs.
no known s. cerevisiae methylation sites documented in swiss-prot contained the rgg, rgx, or rxg-motifs.
however, findmod found  <dig> methylation sites with the rxg or rgx motifs.
two methylation sites, tdh3p dimethyl-r <dig> and rpl4ap dimethyl-r <dig>  matched to the gxxrxg motif, which conforms with the known rxg motif and the additional gxxr motif found in this study.
three methylation sites had the novel wxxxr motif.
a: evidence for the presence of the methylation site on this protein 1: swiss-prot, 2: methylation site confirmed by findmod analysis
b: methylation site matching the specified motif is underlined, the methylation site is highlighted in bold.
discussion
large-scale discovery of lysine and arginine methylation sites
in this study,  <dig> lysine methylation sites and  <dig> arginine methylation sites were identified in  <dig> proteins in the s. cerevisiae proteome.
these include  <dig> proteins previously known to be methylated in yeast or in other organisms and  <dig> proteins that are functionally related to others known to be methylated.
our findings support earlier studies  <cit>  that suggested methylation to be quite widespread.
whilst many of our methylation sites are novel and have not been confirmed by ms-ms, the filters and replicate analyses we used in association with the findmod tool provided a robust means by which protein methylation could be detected.
the false positive rate was estimated to be 11% at  <dig>  da mass error.
notwithstanding this, it should be noted that whilst we did study  <dig>  proteins from yeast, this is only ~40% of the total yeast proteome.
therefore, we expect that up to 60% of methylated proteins would have been missed.
further methylation sites may have been missed due to difficulties in mass spectrometric detection; an example is methylarginine, which is often found in arginine- and glycine-rich regions that produce tryptic peptides that are too small for routine maldi-tof analysis.
discovery rates may reflect the sub-stoichiometric nature of methylation
previous research has highlighted that methylated peptides are difficult to discover  <cit>  and this is made more difficult because methylation is sub-stoichiometric  <cit> .
for example, sub-stoichiometric levels of methylations were observed in the human heterogeneous nuclear ribonucleoprotein k , in which < 33% of hnrnp k were asymmetrically dimethylated at r <dig>  and < 10% were monomethylated at r <dig>  <cit> .
our results from findmod analysis support these observations since the proportion of methylated peptides seen for any protein was very low.
the sub-stoichiometric nature of methylation events was also supported by a weak but significant correlation between the discovery rates of modified and unmodified paired peptides.
however, there may be explanations, other than biological, for the lower discovery rate of modified peptides.
these included inefficient trypsin cleavage which occurs c-terminal to methylated lysine and arginine residues  <cit>  and differences in maldi-tof ionisation of the methylated peptides as seen with different proteotypic peptides  <cit> .
methylated proteins are involved in specific biological functions and processes, are higher in abundance and have longer half-life
methylated proteins were found to be enriched for specific biological processes, molecular functions and sub-cellular localizations.
firstly, methylated proteins were enriched in translation, ribosome biogenesis and assembly.
this is consistent with previous studies in which methylated proteins have been linked to translation in escherichia coli, s. cerevisiae, and schizosaccharomyces pombe  <cit> .
ribosomal proteins are also known to show lysine or arginine methylation, for example the ribosomal proteins l10a, l <dig>  and l26a of arabidopsis  <cit> .
secondly, the methylated proteins described here were found to be involved in rna metabolic processes and are involved in rna binding.
this is consistent with the function of several proteins known to be methylated at rg-rich motifs  <cit> .
the methylation of arginine in rg-rich motifs is conserved in human, and their rna binding activity is also conserved  <cit> .
one such example is the fragile x mental retardation protein   <cit> .
thirdly, our methylated proteins were enriched in the ribosome and the cytoplasm.
this is consistent with the sites of translation and association with rna inside the cell  <cit> .
whilst the lack of methylated proteins enriched in the nucleus and nucleolus was not expected, these may have arisen due to our reduced set of proteins for analysis .
in addition, nuclear proteins such as histone and npl3p are known to have peptides with multiple modification sites but these were not searched for in this study.
methylated proteins found in this study were significantly higher in abundance than proteins currently known to be non-methylated.
this is partly explained by ribosomal proteins and proteins involved in translation, some of which we found to be methylated, being of very high abundance  <cit> .
methylated proteins were also found to be of longer average half-life.
this may be due to their role in translation  <cit> , where ribosomal proteins are generally stable  <cit> .
interplay of methylation and other post-translational modifications
the methylation of lysine is known to block the action of ubiquitin ligase  <cit> , preventing proteins from degradation via the ubiquitin/proteasome system  <cit> .
our observation of a distinct group of low half-life proteins in s. cerevisiae, none of which were methylated, suggests that lysine methylation might be on many proteins and prevent their ubiquitination.
the limited number of ubiquitination sites currently known on yeast proteins  <cit>  makes it currently difficult to check if lysine methylation, as found in this study, is found on residues that can also be poly-ubiquitinated.
however, our prediction of putative ubiquitination sites  <cit>  showed that 43% of the lysine methylation sites in  <dig> proteins may be ubiquitinated.
several studies suggested that there is interplay between arginine methylation and phosphorylation of some proteins  <cit> .
arginine methylation may antagonise phosphorylation  <cit> , act as a switch to enable the binding of phosphatase to encourage dephosphorylation  <cit> , or encourage phosphorylation  <cit> .
on the other hand, phosphorylation can either interfere with arginine methylation  <cit> , or promote the recruitment of arginine methyltransferase  <cit> .
we found that the majority of arginine-methylated proteins in our study  are known from the literature to be phosphorylated, suggesting an interplay between arginine methylation and phosphorylation in these proteins.
however these arginine methylation and phosphorylation sites were not necessarily directly adjacent in the protein sequence.
arginine and lysine methylation motifs
motif analysis showed that many methylation sites described here conform with previously known motifs.
for example,  <dig> arginine methylation sites discovered by findmod conformed with the known rxg and rgx motifs  <cit> .
arginine methylation sites were also enriched in gxxr motifs, which correlated with the enrichment of glycine residues nearby arginine methylation sites  <cit> .
in addition, two experimentally verified methylated sites in pfk2p and rpl23ap annotated in swiss-prot along with  <dig> findmod sites suggests the existence of a mk lysine methylation motif.
the discovery of the novel enriched methylation motif wxxxr supports the possibility that there are more methylation sites to be found in s. cerevisiae.
these also raise an interesting question concerning which motifs are methylated by specific methyltransferases.
methyltransferases responsible for most methylation sites are also unknown , and the function of several methyltransferase proteins in s. cerevisiae remain poorly characterized  <cit> .
therefore, more experiments are required to elucidate the function of methylation in s. cerevisiae.
CONCLUSIONS
this study is a step towards the definition of the methyl proteome of s. cerevisiae.
it will be useful to guide future experiments on its predominance and role in the cell.
for example, experiments are needed to elucidate the function of methylation and how each site is regulated, which with the exception of histone methylation is largely unknown.
secondly, experiments to investigate whether methylation sites overlap with poly-ubiquitination sites, and therefore prevent protein degradation via the ubiquitin/proteasome pathway could be undertaken.
thirdly, it will be important to understand whether the functions of methylated proteins are co-regulated by ubiquitination, phosphorylation or other post-translational modifications.
finally, the ultimate goal in studying methylation should be to build networks of methylated proteins, their interaction partners and modifying enzymes to elucidate their dynamics as a system, similar to previous work on protein phosphorylation  <cit> .

 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. 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 h and histone h. tri-methylation at h3k and h3k is positively correlated with gene activity, while h3k are involved in gene silencing. histone h3k methylation is evolutionarily conserved and is involved in several pathways, including sir protein-mediated heterochromatic gene silencing. meiotic checkpoint control and in the g and s phase dna damage checkpoint functions of rad9p. 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, rpl23p, 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 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+ mrna binding, processing and export, ribosome biogenesis and gene silencing. moreover, methylation is required for the nuclear export of rna binding proteins npl3p, hrp1p, and nab2p. 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, spf and tdrd proteins. in contrast, arginine methylation of sam blocks the interaction of nearby proline-rich motif with an sh domain, but not to a ww domain. more examples on methylarginine-regulated interactions are reviewed in mcbride and silver and bedford and clarke. 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 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, in which methionine was converted to 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. was able to identify methylation on histones from hela cell extracts, such as on histone h3k around 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, hsc 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 protof 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. 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 filters yielded high-confidence methylation sites that were then further investigated to understand where they are found in protein sequences and their likely function.  large-scale methylation discovery in yeast peptide mass spectra findmod was used to analyse peptide mass spectra for yeast proteins out of a total ~ for the presence of mono- and di-methylation. a tailor-made mass tolerance was calculated for each spectrum to reduce spurious peptide matches; the average of this for all spectra was ± da. of all the findmod queries, there were matches to potentially methylated peptides. five filtering strategies, used sequentially, were then applied to this set to find methylation sites of very high confidence. the first filter removed peptides that matched to unmodified peptide sequences as these peptide masses are likely to be unmodified peptides. conversely, peptides masses that did not match to unmodified peptide sequences are likely to be modified, and these were analysed with the second filter. the second filter removed any peptides that contained d or e residues, as artifactual methylation may result from partial methyl esterification of d or e residues. the third filter was designed to take advantage of redundancy within each findmod output, by removing one-off or spurious mass spectra. it searched for modifications that were found in two or more overlapping peptides, and took advantage of the reduced efficiency of tryptic cleavage at methylated residues, where overlapping peptides with missed cleavages were likely to be found. the fourth filter reduced findmod false positives by considering whether modifications found by findmod were unambiguous or ambiguous. an unambiguous modification had only one findmod match against one query peptide mass, an ambiguous modification had more than one match against a query mass. for the peptide to be included in the final set of methylated peptides, at least one peptide in the overlapping peptides had to be an unambiguous peptide match. the use of these filters resulted in high confidence methylated peptides, from initial low confidence matches. while overlapping peptides helped localise methylation sites to one or more peptides, they did not necessarily localise the methylation to one amino acid. to address this, we used a fifth filter. when two or more modified peptides that passed filters 1- were also found to overlap and share the same modification site, the modification was classified as high confidence and kept. note that any results for lysine trimethylation were discarded from the study since it is near-isobaric to lysine acetylation. from this filtering process, we found lysine-methylated proteins with lysine methylation sites: with mono- and with di-methylation. similarly, we found arginine-methylated proteins with arginine methylation sites: with mono- and with di-methylation. there were proteins that contained both arginine and lysine methylation. the list of high confidence methylated proteins and methylation sites are shown in table additional information on these high confidence methylated peptides and methylation sites are shown in additional file and additional file correspondingly. a: m, monomethylated; d, dimethylated confirmation of findmod protein methylation to establish the accuracy of our methylation discovery approach, we theoretically digested all known methylated proteins in swiss-prot and analysed the resulting peptides with our findmod approach. we supplemented this with a larger set of theoretically methylated proteins. the average true positive rate for findmod at da was 89%. for methylation sites in swiss-prot, findmod had a true positive rate of 100% for monomethyl-k, 98% for dimethyl-k, and 76% for dimethyl-r. the true positive rate for monomethyl-r could not be accurately estimated since the number of test cases was insufficient for accurate evaluation. similarly, the true positive rate for the artificial methylation set was 78% for monomethyl-k, 89% for dimethyl-k, and 90% for both monomethyl-r and dimethyl-r. additional results for the evaluation of the true positive rate of findmod are shown in additional file a: n.d. - not determined to further assess the accuracy of the findmod approach, methylation sites discovered by findmod were cross-referenced with known methylation sites in the literature and databases. whilst only a small number of proteins are documented as methylated in the literature, we confirmed proteins as methylated. if we included methyl-lysine sites in peptides containing d and e, we also confirmed the methylation of tef1p and rpl23p. this included lysine methylation sites from tef1p, and lysine dimethylation site from rpl23p. furthermore, we found methylated ribosomal proteins in s. cerevisiae, consistent with the presence of methylation sites in ribosomal proteins of eukaryotes, such as s. cerevisiae, s. pombe, a. thaliana, and human. a: proteins previously known to be methylated in yeast and in other organisms discovery rate of methylated peptides, unmodified peptides, and lysine and arginine-methylated residues the discovery rate of a peptide is the frequency of protein identifications in which a particular peptide is observed. methylated peptides with low discovery rates are likely to be sub-stoichiometric and partially methylated. it was predicted that there should be many more unmodified peptides than methylated peptides, and that methylated peptides will have a lower discovery rate since they are likely to be sub-stoichiometric. the discovery rate of high confidence methylated peptides was found to be significantly lower than that of unmodified peptides. the median discovery rate for unmodified peptides was, and the median value for arginine and lysine methylated peptides was. to check that the lower discovery rate of methylated residues was not due to differences in peptide ionisation efficiency, we examined if there was a correlation between the discovery rates of methylated and unmodified residues. in the set of results, there were methylated residues for which the corresponding unmodified residues were also seen. the discovery rate of methylated residues was significantly but weakly correlated with the discovery rate of matching unmodified residues, consistent with expected. a list of methylated proteins and the methylation sites discovered by findmod is shown in table the discovery rate of all high confidence methylated peptides and methylation sites are shown in additional file and additional file correspondingly. biological function, sub-cellular localization, abundance and half-life of methylated proteins methylated proteins are known to be involved in several pathways, such as translation and rna processing. to investigate the function of the methylated proteins from yeast, gene ontology annotations for all yeast methylated proteins from findmod analysis and swiss-prot were compared to non-methylated yeast proteins. it was found that a number of biological processes were enriched with very high statistical significance, specifically translation, ribosome biogenesis and assembly, rna metabolic process, and organelle organization and biogenesis. the molecular function of structural activity, rna binding, and translation regulator activity were also significantly enriched. as may be expected from the above, methylated proteins were significantly enriched in the cellular components of the ribosome and cytoplasm. a: number of methylated proteins with this go slim term b: number of non-methylated proteins with this go term c: number of methylated proteins with other go slim term d: number of non-methylated proteins with other go slim term protein abundance data from ghaemmaghami et al. was used to compare the abundance of methylated proteins to non-methylated proteins. this was used to determine if lower abundance proteins, more likely to be involved in signal transduction and regulation, are methylated. methylated proteins were found to have a higher median abundance of as compared to non-methylated proteins, which had a median abundance of. despite this, several methylated proteins of low abundance were seen including proteins of less than molecules per cell. these included snf2p, snu114p, mrpl20p, and rpl3p. examples of proteins with high abundance are rp1bp and tdh3p. the methylation of lysine residues has been suggested to block their ubiquitination, leading to a longer protein half-life. to investigate this possibility, protein half-life data from belle et al. was used to compare the half-life of lysine-methylated proteins to non-methylated proteins. interestingly, we found methylated proteins had a longer median half-life of minutes, as compared to minutes for non-methylated proteins. a striking difference between the methylated and non-methylated proteins was the absence of a group of proteins with very short half-life. despite this, our approach also identified methylated proteins with half-life less than minutes. examples of methylated proteins with shorter half-lives are rrp5p, ski3p, and snu114p. examples of proteins with long half-life are utp22p and atp2p, although we note that these numbers may be erroneous estimations in the belle et al. study. although the abundance and half-lives of methylated proteins could be analysed more precisely by comparing methylated proteins to other proteins from the same go slim biological process, this approach was limited by the relatively small number of methylated proteins in the dataset. methylated proteins mapped to gene ontology biological process categories, with an average of proteins per category, which was unsuitable for appropriate statistical analyses. interplay of methylation and other post-translational modifications to see if lysine methylation might block ubiquitination, the ubipred software was used to predict if known methylated lysine sites are also subject to ubiquitination. the ubipred software has an accuracy of % and is thus sufficiently reliable for this test. it was found that 43% of high-confidence lysine methylation sites were also predicted to be ubiquitination sites. this result lends support to the hypothesis that methylation might block ubiquitination, potentially prolonging the half-life of lysine-methylated proteins. it has recently been reported that the methylation of arginine can regulate the phosphorylation of some proteins. to investigate whether there is evidence of interplay between arginine methylation and phosphorylation in s. cerevisiae, we examined the proportion of arginine-methylated proteins that are known to be phosphorylated in databases and in the literature. it was found that 94% of arginine-methylated yeast proteins are known to be phosphorylated. this is a considerable increase over the 38% of all s. cerevisiae proteins known to be phosphorylated and suggests a possible interplay of arginine methylation and phosphorylation. arginine and lysine methylation motifs to determine if methylation sites are enriched in specific sequence-motifs, all yeast methylation sites from findmod analysis and the swiss-prot database were analysed to find enriched sequence-motifs. methionine was found to be at position - from lysine methylation in findmod sites and two additional methylation sites previously documented in s. cerevisiae. this presence of methylation was of very high statistical significance as compared to that expected in any random sequence of yeast proteins. by contrast, residues found to be significantly enriched adjacent to arginine methylation included w at position -, and g at position -. while it was previously known that arginine methylation is found in rgg motifs, wooderchak et al. showed that arginine methylation is also found in rxg and rgx motifs. no known s. cerevisiae methylation sites documented in swiss-prot contained the rgg, rgx, or rxg-motifs. however, findmod found methylation sites with the rxg or rgx motifs. two methylation sites, tdh3p dimethyl-r and rpl4ap dimethyl-r matched to the gxxrxg motif, which conforms with the known rxg motif and the additional gxxr motif found in this study. three methylation sites had the novel wxxxr motif. a: evidence for the presence of the methylation site on this protein 1: swiss-prot, 2: methylation site confirmed by findmod analysis b: methylation site matching the specified motif is underlined, the methylation site is highlighted in bold. discussion large-scale discovery of lysine and arginine methylation sites in this study, lysine methylation sites and arginine methylation sites were identified in proteins in the s. cerevisiae proteome. these include proteins previously known to be methylated in yeast or in other organisms and proteins that are functionally related to others known to be methylated. our findings support earlier studies that suggested methylation to be quite widespread. whilst many of our methylation sites are novel and have not been confirmed by ms-ms, the filters and replicate analyses we used in association with the findmod tool provided a robust means by which protein methylation could be detected. the false positive rate was estimated to be 11% at da mass error. notwithstanding this, it should be noted that whilst we did study proteins from yeast, this is only ~40% of the total yeast proteome. therefore, we expect that up to 60% of methylated proteins would have been missed. further methylation sites may have been missed due to difficulties in mass spectrometric detection; an example is methylarginine, which is often found in arginine- and glycine-rich regions that produce tryptic peptides that are too small for routine maldi-tof analysis. discovery rates may reflect the sub-stoichiometric nature of methylation previous research has highlighted that methylated peptides are difficult to discover and this is made more difficult because methylation is sub-stoichiometric. for example, sub-stoichiometric levels of methylations were observed in the human heterogeneous nuclear ribonucleoprotein k, in which < 33% of hnrnp k were asymmetrically dimethylated at r and < 10% were monomethylated at r. our results from findmod analysis support these observations since the proportion of methylated peptides seen for any protein was very low. the sub-stoichiometric nature of methylation events was also supported by a weak but significant correlation between the discovery rates of modified and unmodified paired peptides. however, there may be explanations, other than biological, for the lower discovery rate of modified peptides. these included inefficient trypsin cleavage which occurs c-terminal to methylated lysine and arginine residues and differences in maldi-tof ionisation of the methylated peptides as seen with different proteotypic peptides. methylated proteins are involved in specific biological functions and processes, are higher in abundance and have longer half-life methylated proteins were found to be enriched for specific biological processes, molecular functions and sub-cellular localizations. firstly, methylated proteins were enriched in translation, ribosome biogenesis and assembly. this is consistent with previous studies in which methylated proteins have been linked to translation in escherichia coli, s. cerevisiae, and schizosaccharomyces pombe. ribosomal proteins are also known to show lysine or arginine methylation, for example the ribosomal proteins l10a, l and l26a of arabidopsis. secondly, the methylated proteins described here were found to be involved in rna metabolic processes and are involved in rna binding. this is consistent with the function of several proteins known to be methylated at rg-rich motifs. the methylation of arginine in rg-rich motifs is conserved in human, and their rna binding activity is also conserved. one such example is the fragile x mental retardation protein. thirdly, our methylated proteins were enriched in the ribosome and the cytoplasm. this is consistent with the sites of translation and association with rna inside the cell. whilst the lack of methylated proteins enriched in the nucleus and nucleolus was not expected, these may have arisen due to our reduced set of proteins for analysis. in addition, nuclear proteins such as histone and npl3p are known to have peptides with multiple modification sites but these were not searched for in this study. methylated proteins found in this study were significantly higher in abundance than proteins currently known to be non-methylated. this is partly explained by ribosomal proteins and proteins involved in translation, some of which we found to be methylated, being of very high abundance. methylated proteins were also found to be of longer average half-life. this may be due to their role in translation, where ribosomal proteins are generally stable. interplay of methylation and other post-translational modifications the methylation of lysine is known to block the action of ubiquitin ligase, preventing proteins from degradation via the ubiquitin/proteasome system. our observation of a distinct group of low half-life proteins in s. cerevisiae, none of which were methylated, suggests that lysine methylation might be on many proteins and prevent their ubiquitination. the limited number of ubiquitination sites currently known on yeast proteins makes it currently difficult to check if lysine methylation, as found in this study, is found on residues that can also be poly-ubiquitinated. however, our prediction of putative ubiquitination sites showed that 43% of the lysine methylation sites in proteins may be ubiquitinated. several studies suggested that there is interplay between arginine methylation and phosphorylation of some proteins. arginine methylation may antagonise phosphorylation, act as a switch to enable the binding of phosphatase to encourage dephosphorylation, or encourage phosphorylation. on the other hand, phosphorylation can either interfere with arginine methylation, or promote the recruitment of arginine methyltransferase. we found that the majority of arginine-methylated proteins in our study are known from the literature to be phosphorylated, suggesting an interplay between arginine methylation and phosphorylation in these proteins. however these arginine methylation and phosphorylation sites were not necessarily directly adjacent in the protein sequence. arginine and lysine methylation motifs motif analysis showed that many methylation sites described here conform with previously known motifs. for example, arginine methylation sites discovered by findmod conformed with the known rxg and rgx motifs. arginine methylation sites were also enriched in gxxr motifs, which correlated with the enrichment of glycine residues nearby arginine methylation sites. in addition, two experimentally verified methylated sites in pfk2p and rpl23ap annotated in swiss-prot along with findmod sites suggests the existence of a mk lysine methylation motif. the discovery of the novel enriched methylation motif wxxxr supports the possibility that there are more methylation sites to be found in s. cerevisiae. these also raise an interesting question concerning which motifs are methylated by specific methyltransferases. methyltransferases responsible for most methylation sites are also unknown, and the function of several methyltransferase proteins in s. cerevisiae remain poorly characterized. therefore, more experiments are required to elucidate the function of methylation in s. cerevisiae.  this study is a step towards the definition of the methyl proteome of s. cerevisiae. it will be useful to guide future experiments on its predominance and role in the cell. for example, experiments are needed to elucidate the function of methylation and how each site is regulated, which with the exception of histone methylation is largely unknown. secondly, experiments to investigate whether methylation sites overlap with poly-ubiquitination sites, and therefore prevent protein degradation via the ubiquitin/proteasome pathway could be undertaken. thirdly, it will be important to understand whether the functions of methylated proteins are co-regulated by ubiquitination, phosphorylation or other post-translational modifications. finally, the ultimate goal in studying methylation should be to build networks of methylated proteins, their interaction partners and modifying enzymes to elucidate their dynamics as a system, similar to previous work on protein phosphorylation. 
in this study, we examined previously generated peptide mass spectra from saccharomyces cerevisiae proteins for the presence of arginine and lysine methylation. functionally, methylated proteins were significantly enriched for protein translation, ribosomal biogenesis and assembly and organellar organisation and were predominantly found in the cytoplasm and ribosome. this was done using the findmod tool and filters that took advantage of the high number of replicate analysis per protein and the presence of overlapping peptides. some 43% of methylated lysine sites were predicted to be amenable to ubiquitination, suggesting methyl-lysine might block the action of ubiquitin ligase. a total of high-confidence lysine and arginine methylation sites were found in proteins.
 the methylation of eukaryotic proteins has been proposed to be widespread, but this has not been conclusively shown to date. in this study, we examined previously generated peptide mass spectra from saccharomyces cerevisiae proteins for the presence of arginine and lysine methylation. this was done using the findmod tool and filters that took advantage of the high number of replicate analysis per protein and the presence of overlapping peptides.  a total of high-confidence lysine and arginine methylation sites were found in proteins. motif analysis revealed many methylated sites were associated with mk, rgg/rxg/rgx or wxxxr motifs. functionally, methylated proteins were significantly enriched for protein translation, ribosomal biogenesis and assembly and organellar organisation and were predominantly found in the cytoplasm and ribosome. intriguingly, methylated proteins were seen to have significantly longer half-life than proteins for which no methylation was found. some 43% of methylated lysine sites were predicted to be amenable to ubiquitination, suggesting methyl-lysine might block the action of ubiquitin ligase.  this study suggests protein methylation to be quite widespread, albeit associated with specific functions. large-scale tandem mass spectroscopy analyses will help to further confirm the modifications reported here. 