Peptide Sequence Influence on the Differentiation of Valine and Norvaline by Hot Electron Capture Dissociation

Isomeric amino acid residues such as valine (Val) and norvaline (Nva) are common in recombinant proteins. The mis-incorporation of Nva for leucine (Leu) causes heterogeneity and in some cases even toxicity. Previous studies have shown that hot electron capture dissociation (HECD) is able to differentiate Val from Nva by producing diagnostic w ions on custom designed synthetic model peptides. To broaden the utilization of HECD in proteomic studies and to define the critical structural features, a thorough investigation was performed on representative peptides including specifically designed synthetic peptides as well as biological peptides bearing tryptic digest-like features and peptides with post-translational modifications. Experimental evidence confirmed that the formation of a w ion is directly dependent upon the presence of the corresponding z ion. The results suggested that a charge carrier residue at the C-terminus is promoting the formation of diagnostic w ions for Nva. Thus, peptides resulting from trypsin digestion, with arginine (Arg) or lysine (Lys) at the C-terminus, can be analyzed using the HECD method. Post-translational modification (PTM) such as phosphorylation did not prevent the generation of the requisite side chain fragmentation w ions. These results suggest the general applicability of HECD for unambiguous identification of Val and Nva especially in structure characterization of therapeutic proteins.