Quantitation and localisation of (in vitro) transglutaminase-catalysed glutamine hydroxylation using mass spectrometry

A mass spectrometric approach was chosen to quantify and localise in vitro enzymatically modified glutamine (Gln) residues in a glutamine-rich protein. This protein (named dB1), a cloned domain of the high molecular weight wheat glutenin subunit Dx5, was modified by microbial transglutaminase (TGase) using hydroxylamine as the amine donor. Using MALDI-TOF MS (matrix assisted laser desorption ionization-time of flight detection mass spectrometry) it was found that maximally 70% of the 64 Gln residues of dB1 were modified after prolonged incubation with TGase and hydroxylamine. Next, modified dB1 was proteolytically digested and the peptides obtained were subjected to nanospray MS/MS analysis. Using this analysis, the peptides could be identified, and in a second stage of the analysis, a number of modified Gln residues were localised. The results show that indeed some of the Gln residues in dB1 can not be modified by TGase, and that these non-modified Gln are flanked C-terminally by a proline residue. The analysis method described in this article is generic and can be applied to other (in vitro) protein modification products as well.