DIFFERENTIATION OF ISOMERIC AMINO ACID RESIDUES IN PEPTIDES USING ECD

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Under physiological conditions, asparagine (Asn) residues can deamidate spontaneously, generating a mixture of aspartic (Asp) and isoaspartic acid (isoAsp) residues via a succinimide intermediate. In addition, isoAsp residue may also be formed through Asp isomerization, although this occurs at a much slower rate. Differentiation of Asp and isoAsp residues is important as the latter often causes more significant changes in protein conformation and functions, which has been linked to many protein misfolding diseases and other pathological processes. An electron capture dissociation (ECD)-MS/MS based method was established in this laboratory, where diagnostic c+57/z+-57 ions from isoAsp residues were used for the differentiation and relative quantification of the two isomeric forms. This method has been applied to study the Asn deamidation and Asp isomerization in several model systems below. (1) Development of a top-down approach for isoaspartomics research: (2) Use of the endoprotease Glu-C in bottom-up isoAsp analysis: (3) N-terminal isoAsp identification: (4) Differentiation of glutamine deamidation products using ECD and ETD: This research has been published in Analytical Chemistry, 2010, 82, 3606-3615. (5) Unusual fragmentation of beta-linked peptides by ECD and ETD: This research has been published in the Journal of the American Society for Mass Spectrometry, 2011, 22, 480-491.