Abstract
Purpose :
Deamidation, a major aged-related modification in lens, is difficult to study, because it causes a nominal 1 Da mass increase. However, the 19-mDa mass difference between isotopic peaks of deamidated and non-deamidated peptides can be fully resolved with a high-resolution mass spectrometer. This methodology was used to test whether deamidation in crystallins was more extensive in lens water-insoluble fractions.
Methods :
The nuclear region of 85 and 88-year old human donors with differences in brunescence were dissected, proteins fractioned by centrifugation at 16,000 x g into water-soluble and insoluble fractions, suspended in 8M urea, then digested with trypsin. Soluble protein from a 5-day old human lens was used as a control. 250 ng of each digest was then separated on a 25 cm nanospray UPLC C18 column (4-hour gradient) and analyzed using an Orbitrap Fusion mass spectrometer. Survey MS scans were acquired at 500,000 resolution, while data-independent ion trap MS/MS scans were acquired over the entire mass range in 12 m/z increments. Chromatograms for peptides were extracted and peaks integrated using Skyline software.
Results :
Chromatograms for deamidated and non-deamidated peptide forms were produced with no interference, while simultaneously monitoring fragment ions to confirm identities. The utility of this methodology was demonstrated by measuring the relative extent of deamidation in γS-crystallin peptides 7-18, 72-78, and 131-145. The extent of deamidation in these peptides from 5 day-old lens was 1.4, 3.6, and 1.7%, respectively, and increased to 38, 83, and 61% in the 85-year old lens insoluble fraction. These values averaged 18% lower in the 88-year-old lens with less brunescence. The fold increases in % deamidation between the water-insoluble and soluble fractions for these peptides in the 85-year-old lens were 3.2, 5.8, and 6.6, respectively.
Conclusions :
The results show that a combination of high-resolution survey scans and data-independent fragment ion acquisition greatly improve the detection and quantification of deamidation. Data supports earlier findings that deamidation in γS is found primarily in the lens water-insoluble fraction, and that it may be a cause of crystallin insolubilization with age.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.