Preparation of samples for MS analysis

In vitro methylation of recombinant or cellular (in extract) proteins, for the purpose of mass spectrometry (MS) analysis, was performed as in the above section, except that [³H]AdoMet was replaced with non-radioactive AdoMet (1 mM). Proteins were resolved by SDS-PAGE, stained with Coomassie Blue, and the portion of gel containing the protein of interest was excised and subjected to in-gel trypsin (Sigma-Aldrich), GluC (Promega), chymotrypsin or AspN (Roche) digestion, and the resulting proteolytic fragments were analyzed by liquid chromatography (LC) MS. Reversed phase (C18) LC was performed using the Dionex Ultimate 3000 UHPLC systems (Thermo Fisher Scientific). The peptide solution (5 μl) was injected into the extraction μ-Precolumn (Acclaim PepMap100, C18, 5 μm resin, 100 Å, 300 μm i.d. × 5 mm) (Thermo Fisher Scientific) and peptides were eluted in back-flush mode onto the analytical column (Acclaim PepMap100, C18, 3 μm resin, 100 Å, 75 μm i.d. × 5 cm, nanoViper) (Thermo Fisher Scientific). The mobile phase consisted of acetonitrile and MS grade water, both containing 0.1% formic acid. Chromatographic separation was achieved using a binary gradient from 3 to 50% of acetonitrile in water for 60 min, with 0.3 μl/min flow rate. The LC system was coupled via a nano-electrospray ion source to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Peptide samples were analyzed with a high energy collisional dissociation fragmentation method with normalized collision energy set at 28, acquiring one Orbitrap survey scan in the mass range of m/z 300–2000, followed by MS/MS of the ten most intense ions in the Orbitrap.

MS data were analyzed with in-house maintained human, rat and mouse protein sequence databases using SEQUEST™ and Proteome Discoverer™ (Thermo Fisher Scientific). The mass tolerances of a fragment ion and a parent ion were set as 0.5 Da and 10 ppm, respectively. Methionine oxidation and cysteine carbamido-methylation were selected as variable modifications. MS/MS spectra of peptides corresponding to methylated eEF1A1 and eEF1A2 were manually searched by Qual Browser (v2.0.7).