Fig. 2
Identification of the Δ−16 Da modification as O-methyl-l-homoserine (methoxine) at methionine residues. a Tryptic digest of Nanobody B, produced and purified from the CBS7435MutS strain, generated 17 peptides. Retention times on RP-HPLC are indicated. Small peptides eluted in the flow through (FT). Four additional peptides were detected with a mass difference of −16 Da and showed a shorter retention time on RP-HPLC than their corresponding counterparts (peptides 2, 8, 10 and 16). These four peptides all contained a methionine residue. MS/MS fragmentation of the four peptides showed that the Δ−16 Da modification was located at the methionine (see Additional file 1: Figure S1); b and c Two possible amino acid substitutions resulting in a mass difference of Δ−16 Da were plausible: methionine → aspartic acid and methionine → O-methyl-l-homoserine. Two synthetic peptides containing these substitutions were made and their behaviour compared on RP-HPLC to the corresponding Δ−16 Da of peptide ten that was generated after trypsin digest of Nanobody B. The peptide with incorporation of O-methyl-l-homoserine elutes at the same retention time of the peptide that contains the Δ−16 Da modification (28.4 min) whereas the peptide containing the aspartic acid substitution showed a shorter retention time of 25.0 min (c). * Aspecific tryptic cleavage product of peptide 8 (see Additional file 1: Figure S1C, D); ** unknown peptide