FIGURE 3.

Deletion of EFM3 results in loss of trimethyllysine 509 on elongation factor 2. The 100-kDa protein bands from wild type and knock-out lysates were excised, in-gel trypsin-digested, and analyzed by LC-MS/MS. A Mascot search identified EF2 as the top hit (score = 13,763.99) for the 100 kDa band. Known trimethylation at Lys-509 (TMK509) (A) and dimethylation at Lys-613 (DMK613) (B) were identified in EF2 peptides LVEGLKR and DDFKAR, respectively, in wild type lysate. Dimethylation at Lys-509 (DMK509) and monomethylation at Lys-613 (MMK613) were also observed in EF2 from wild type lysate. C, the effect of efm2Δ and efm3Δ on the methylation of Lys-509 and Lys-613 was examined by comparison of EF2 from wild-type lysate with that from knock-out lysates. PRM-MS with extracted precursor → product ion transitions (within 10 ppm) was used to identify methylated peptides in each sample condition (WT, efm2Δ, and efm3Δ).