Characterization of Glutamine Deamidation by Long-Length Electrostatic Repulsion-Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry (LERLIC-MS/MS) in Shotgun Proteomics
Sze, Siu Kwan
Date of Issue2016
School of Biological Sciences
Deamidation of glutamine (Gln) residues is a spontaneous or enzymatic process with significant implications in aging and human pathology. Although some methods are available to identify the γ/α-glutamyl products of deamidation, none of these methods allows the characterization of this post-translational modification (PTM) from complex biological samples by shotgun proteomics. Here we present LERLIC-MS/MS, a chromatographic strategy that uses a long (50 cm) anion-exchange capillary column operating in the electrostatic repulsion-hydrophilic interaction mode (ERLIC) and coupled directly to tandem mass spectrometry (MS/MS) for proteome analysis in a single injection. Profiling of soluble extracts of brain tissues by LERLIC-MS/MS distinguished for the first time γ/α-glutamyl isomers of deamidation, encountering a 1.7 γ/α-glutamyl ratio for most Gln deamidation products. A detailed analysis of any deviation from that observed ratio allowed the identification of transglutaminase-mediated γ-glutamyl isomers as intermediate products of transamidation. Furthermore, LERLIC-MS/MS was able to simultaneously separate Gln and asparagine (Asn) deamidation products even for those peptides showing multiple deamidated proteoforms. The characterization of Asn deamidated residues by LERLIC-MS/MS also uncovered novel PIMT (protein L-isoaspartyl methyltransferase) substrate proteins in human brain tissues that deviated from the expected 3:1 isoAsp/Asp ratio. Taken together, our results demonstrate that LERLIC-MS/MS can be used to perform an in-depth study of protein deamidation on a global proteome scale. This new strategy should help to elucidate the biological implications of deamidation in aging and disease conditions.
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