Glutamine Deamidation: Differentiation of Glutamic Acid and γ-Glutamic Acid in Peptides by Electron Capture Dissociation

Abstract

Due to its much slower deamidation rate compared to that of asparagine (Asn), studies on glutamine (Gln) deamidation have been scarce, especially on the differentiation of its isomeric deamidation products: α- and γ-glutamic acid (Glu). It has been shown previously that electron capture dissociation (ECD) can be used to generate diagnostic ions for the deamidation products of Asn: aspartic acid (Asp) and isoaspartic acid (isoAsp). The current study explores the possibility of an extension of this ECD based method to the differentiation of the α- and γ-Glu residues, using three human Crystallin peptides (αA (1-11), βB2 (4-14), and γS (52-71)) and their potentially deamidated forms as model peptides. It was found that the z^•-72 ions can be used to both identify the existence and locate the position of the γ-Glu residues. When the peptide contains a charge carrier near its N-terminus, the c+57 and c+59 ions may also be generated at the γ-Glu residue. It was unclear whether formation of these N-terminal diagnostic ions is specific to the Pro-γ-Glu sequence. Unlike the Asp containing peptides, the Glu containing peptides generally do not produce diagnostic side chain loss ions, due to the instability of the resulting radical. The presence of Glu residue(s) may be inferred from the observation of a series of z_n^•-59 ions, although it was neither site specific nor without interference from the γ-Glu residues. Finally, several interference peaks exist in the ECD spectra, which highlights the importance of the use of high performance mass spectrometers for confident identification of γ-Glu residues.