The lens contains some of the oldest proteins in the body that undergo deamidation, isomerization, and racemization at asparagine and aspartic acids during cataract. This study identifies these modifications in a major lens protein called ɣS-crystallin using stable isotope-labeled peptides, and confidently assigns deamidation status using high-resolution mass spectrometry.

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 identify 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 in characterizing deamidation posttranslational modifications. A total of 32 stable isotope-labeled peptides are 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 is used to accurately assign deamidation status using the 19 mDa mass defect between isotopic peaks of deamidated and nondeamidated peptides. While peptides containing D-forms of Asp and isoAsp were assigned, the predominant isoforms contained L-isoAsp. High-resolution mass spectrometry using wide single ion monitoring data-independent acquisition 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.