April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Identification Of Crystallin Disulfide Cross-Links In Cataractous Human Lens By Electron Transfer Dissociation
Author Affiliations & Notes
  • Larry L. David
    Biochemistry & Molecular Biology, Oregon Health & Science Univ, Portland, Oregon
  • Phillip A. Wilmarth
    Biochemistry & Molecular Biology, Oregon Health & Science Univ, Portland, Oregon
  • Footnotes
    Commercial Relationships  Larry L. David, None; Phillip A. Wilmarth, None
  • Footnotes
    Support  NIH grants EY010572 and EY007755
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2083. doi:
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      Larry L. David, Phillip A. Wilmarth; Identification Of Crystallin Disulfide Cross-Links In Cataractous Human Lens By Electron Transfer Dissociation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2083.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Disulfide cross-linked crystallins may be a major cause of light scatter in age-related nuclear cataract. While analysis of intermolecular disulfide cross-links in crystallins would provide important information on the structure of light scattering aggregates, the identification of the individual peptides participating in cross-links is problematic, because fragmentation from both peptides occurs simultaneously during tandem mass spectrometry, resulting in spectra that are difficult to interpret. The purpose of this study was to demonstrate the utility of electron transfer dissociation (ETD) as a tool to simplify the analysis of lens disulfide cross-links.

Methods: : Water-insoluble protein from a 93-year-old human lens with age-related nuclear cataract and an 82-year-old normal lens were alkylated with iodoacetamide without prior reduction, digested with trypsin, and peptides separated into 34 fractions by cation-exchange chromatography. Each fraction was then separated by reverse-phase chromatography and tandem mass spectrometry data acquired using an LTQ Velos linear ion trap with an ETD source (Thermo Scientific). Each survey MS scan was alternated with a single data-dependant MS2 scan using ETD to break the disulfide bond between cross-linked peptides, followed by 5 MS3 scans where individual peptides liberated by ETD were sequenced using standard collision-induced dissociation. Disulfide cross-linked peptides were then identified in these linked MS3 scans using Sequest.

Results: : Disulfide cross-linked peptides from crystallins were efficiently fragmented at disulfide bonds by ETD to yield individual peptides that were then identified by a standard tandem MS technique and database searches. The majority of disulfide bonds identified in the insoluble fraction of cataractous lens were potentially intramolecular, since they occurred between cysteine containing peptides within a single crystallin subunit. However, intermolecular disulfide bonded peptides were also observed, such as between peptide 163-177 in βA3-crystallin and peptide 126-131 of γS-crystallin.

Conclusions: : Combining a multiple-dimensional liquid chromatography/mass spectrometry approach and ETD to specifically break disulfide bonds in the insoluble protein of cataractous lenses is a powerful technique to identify major disulfide cross-links in crystallins. These results will provide new information about the structure of light scattering aggregates in cataractous lens.

Keywords: crystallins • proteomics • cataract 

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