To assign hmSILAC peptide sequences, we defined new modifications in MaxQuant with the mass increment and residue specificities corresponding to heavy monomethylation (mono-methyl4-K/R) and dimethylation (di-methyl4-K/R). In addition, we defined new modifications for heavy methionine (Met4) and oxidized heavy methionine (OxMet4). To reduce the search complexity, raw data were analyzed twice with the following sets of variable modifications: (i) N-terminal acetylation, Met4, OxMet4, oxidation, mono-methyl-K/R, mono-methyl4-K/R; (ii) N-terminal acetylation, Met4, OxMet4, oxidation, di-methyl-K/R, di-methyl4-K/R.

Identification of high-confidence methyl sites was carried out with an in-house developed, Perl-based pipeline, named hmSEEKER (36). hmSEEKER identifies doublets of heavy and light hmSILAC peptides from MaxQuant output tables. However, during data-dependent acquisition, the choice of peptide ions to fragment is stochastic, so there are no guarantees that the heavy and light forms will both be sequenced. Hence, we adopted a strategy to retrieve heavy/light counterparts that are not identified by MS/MS. Briefly, the doublet search step is performed as follows: Information regarding each peptide from the evidence.txt MaxQuant output file is stored in an indexed table. Similarly, MS1 peak information is extracted from the msmsScans.txt file and stored in a second index. Each peptide from the first index is then associated to its corresponding peak in the second one, in which the search of corresponding heavy/light counterpart is performed. This retrieves a counterpart and builds an H/L methyl peptide pair even when one of the two is not MS/MS-sequenced but only detected as a peak in the MS1 spectrum. The doublets search is based on the assumption that heavy and light counterparts of the same modified peptide co-elute from the chromatographic column and differ for a highly specific, unambiguously associated delta mass, which we can measure at high resolution in MS1. We used hmSEEKER to automatically filter the MaxQuant evidence.txt file and remove contaminants and reverse sequences, as well as peptides carrying simultaneously light and heavy modifications. To increase the confidence of our findings, the remaining peptides were further filtered to remove any peptide with Andromeda score < 25 or Andromeda delta score < 12 and any methylation with a PTM localization probability < 0.75. Heavy and light methyl peptide pairs were considered true positive when the difference between calculated and expected mass shift was <2 ppm and the difference between their retention times was <30 s.

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