Identification of peptide isoforms in gS-crystallin using stable isotope labeled peptides

ɣS-crystallin is a major protein of the human lens and is highly modified with age and cataract due to a lack of lens protein turnover. Previous studies have identified some sites of isomerization and racemization of deamidated asparaginyl and aspartyl residues in ɣS but have been limited due to the complexity of isoforms and difficulty characterizing deamidation post-translational modifications. A total of 32 stable isotope labeled peptides were created for ɣS residues 7-18, 72-78, and 131-145 containing L-Asp, D-Asp, L-isoAsp, and D-isoAsp at D12, N14, N76, D77, and N143 to act as internal chromatography standards spiked into tryptic digests of nuclear insoluble protein of a cataractous human lens. High-resolution mass spectrometry was used to accurately assign deamidation status using the 19 mDa mass defect between isotopic peaks of deamidated and non-deamidated peptides. While peptides containing D-forms of Asp and isoAsp were assigned, the predominate isoforms contained L-isoAsp. High-resolution mass spectrometry using wide single ion monitoring- data-independent acquisition (WiSIM-DIA) also greatly improved the reliable identification of peptide deamidation states. These results will aid creation of ɣS using native chemical ligation to examine the role of isoAsp in crystallin aggregation and cataract.