As post-translational modification (PTM) of proteins broadly regulates their biological function and activity, specific modifications are likely useful biomarkers. The prevailing approach to PTM characterization has been MS/MS and particularly electron capture or transfer dissociation (EC/TD), but commonly substoichiometric and weak PTM attachment causes major specificity and sensitivity challenges, especially when multiple actual or potential localization variants exist. We report that variant phosphopeptides can be fully separated and identified employing ion mobility spectrometry (IMS), either conventional IMS or differential IMS (FAIMS). These include isomers with multiple modifications, serine and threonine phosphorylations, and alternative modifications of adjacent sites, which are most difficult to distinguish by MS/MS. Avoiding the MS/MS step permits improving sensitivity by >10 times compared to ETD on an Orbitrap, while maintaining linear quantification. The results are illustrated using model peptides and tryptic sequences from the human tau-protein, where variant phosphorylation is believed relevant to the Alzheimer's disease.

Detection of post-translationally modified peptide biomarkers using ion mobility spectrometry platforms

242nd National Meeting (2011)

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