Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS. (February 2016)

• Record Type: Journal Article
• Authors: Gahoual, Rabah; Beck, Alain; François, Yannis‐Nicolas; Leize‐Wagner, Emmanuelle
• Is Part Of: Journal of mass spectrometry. Volume 51:Number 2(2016)
• Page Start: 150
• Page End: 158
• Publication Date: 2016-02
• Subjects: electrospray ionization mass spectrometry -- capillary electrophoresis -- post‐translational modifications -- asparagine deamidation -- aspartic acid isomerization -- monoclonal antibody structure characterization -- bottom‐up proteomics analysis; Mass spectrometry -- Periodicals; 543.65
• DOI: 10.1002/jms.3735
• Languages: English
• ISSNs: 1076-5174

Abstract

Abstract : Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation ofl ‐aspartic acid ord ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd.