Separation of Peptide Isomers with Variant Modified Sites by High-resolution Differential Ion Mobility Spectrometry

Overview

Journal Anal Chem

Specialty Chemistry

Date 2010 Sep 17

PMID 20843012

Citations 39

Authors

Alexandre A Shvartsburg

Andrew J Creese

Richard D Smith

Helen J Cooper

Affiliations

Soon will be listed here.

Abstract

Many proteins and proteolytic peptides incorporate the same post-translational modification (PTM) at different sites, creating multiple localization variants with different functions or activities that may coexist in cells. Current analytical methods based on liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS) are challenged by such isomers that often coelute in LC and/or produce nonunique fragment ions. The application of ion mobility spectrometry (IMS) was explored, but success has been limited by insufficient resolution. We show that high-resolution differential ion mobility spectrometry (FAIMS) employing helium-rich gases can readily separate phosphopeptides with variant modification sites. Use of He/N(2) mixtures containing up to 74% He has allowed separating to >95% three monophosphorylated peptides of identical sequence. Similar separation was achieved at 50% He, using an elevated electric field. Bisphosphorylated isomers that differ in only one modification site were separated to the same extent. We anticipate FAIMS capabilities for such separations to extend to other PTMs.

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