Isomerized and Racemized Aspartyl and Deamidated Asparagine Residues Identified in ɣS-Crystallin

Area of Science:

• Biochemistry
• Proteomics
• Ocular Biology

Background:

• ɣS-crystallin, a key human lens protein, undergoes age-related modifications like deamidation due to limited protein turnover.
• Previous characterization of ɣS-crystallin deamidation was hindered by complex isoforms and analytical challenges.

Purpose of the Study:

• To accurately characterize deamidation post-translational modifications in ɣS-crystallin.
• To develop and apply methods for identifying specific deamidated residues and their stereoisomeric forms.

Main Methods:

• Synthesis of 32 stable isotope-labeled peptides representing ɣS-crystallin residues with various L-Asp, D-Asp, L-isoAsp, and D-isoAsp forms.
• Analysis of tryptic digests from cataractous human lens nuclear insoluble protein using high-resolution mass spectrometry.
• Utilizing the 19 mDa mass defect for accurate deamidation status assignment and employing wide single ion monitoring data-independent acquisition.

Main Results:

• Successfully assigned deamidation status for peptides containing D-Asp and isoAsp forms.
• Identified L-isoaspartate as the predominant deamidated isoform in cataractous human lens ɣS-crystallin.
• Demonstrated improved reliability in identifying peptide deamidation states via high-resolution mass spectrometry.

Conclusions:

• Established a robust method for characterizing ɣS-crystallin deamidation using mass spectrometry.
• The findings highlight L-isoaspartate as a significant modification in age-related lens changes.
• Results provide a foundation for future studies on isoAsp's role in crystallin aggregation and cataract formation using native chemical ligation.