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Larry David, Eileen Yue, Phillip Wilmarth; A Targeted Proteomics Approach Using Stable Isotope labeling To Measure Sulfhydryl Oxidation In Human Lens Crystallins. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2972. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To simplify the measurement of disulfide bonding in human lens crystalins to determine which cysteines are most susceptible to oxidation during cataract.
Water-insoluble protein from the nucleus of 82, 78, 85, 88, and 93-year-old human donors was isolated from lenses with grades of nuclear opacity between 0-4, respectively. Free sulfhydryls were then alkylated with iodoacetic acid, followed by reduction, and alkylation of reduced disulfides with a heavy isotope form of iodoacetic acid containing two 13C and two 18O atoms. Following trypsinization, the relative abundance of both light and heavy forms of 30 different cysteine containing crystallin peptides were measured using single reaction monitoring (SRM) on a LTQ Velos Pro ion trap mass spectrometer by scheduling 60 transitions during a single 140 min LC-MS run.
The % disulfide involvement at most cysteine residues was remarkably well correlated with the extent of opacity and ranged from 4-87% when going from an opacity score of 0-4. However, several β-crystallin peptides were relatively resistant to oxidation until severe opacity formed, such as the two cysteines in βB2, and single cysteine in βB1. In contrast, many γ-crystallin cysteines were 40% or more oxidized in the 82-year old lens without cataract. The most oxidized cysteines in normal lens were in γ-crystallin peptides containing multiple cysteines.
The use of stable isotope labeled iodoacetic acid coupled with targeted data collection by SRM greatly simplified sulfhydryl oxidation measurements in lens by avoiding the need for expensive reagents or lengthy 2D-LC/MS runs. The addition of unlabeled iodoacetic acid when lens proteins are extracted will determine if disulfides found in normal aged lens are artifactually created. Formation of disulfide bonds between adjacent cysteines may be the first indicator of oxidative stress in lens.
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