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Characterization of Glutamine Deamidation by Long-Length Electrostatic Repulsion-Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry (LERLIC-MS/MS) in Shotgun Proteomics
journal contribution
posted on 2016-09-30, 00:00 authored by Aida Serra, Xavier Gallart-Palau, Juan Wei, Siu Kwan SzeDeamidation 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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Asn deamidated residuesratiotandem mass spectrometryPTMbrain tissuesRepulsion-Hydrophilic Interaction Chromatography-Tandem Mass Spectrometryrepulsion-hydrophilic interaction modeLERLIC-MSMStransglutaminase-mediated γ- glutamyl isomersGln deamidation productsanion-exchange capillary columnPIMTERLICShotgun Proteomics Deamidationprotein L-isoaspartyl methyltransferase