March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Thioether Modifications at Phosphorylation Sites in Aged Human Lens Proteins
Author Affiliations & Notes
  • Kevin L. Schey
    Biochemistry, Vanderbilt University, Nashville, Tennessee
  • Zhen Wang
    Biochemistry, Vanderbilt University, Nashville, Tennessee
  • Footnotes
    Commercial Relationships  Kevin L. Schey, None; Zhen Wang, None
  • Footnotes
    Support  EY13462; P30 EY08126
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1302. doi:
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      Kevin L. Schey, Zhen Wang; Thioether Modifications at Phosphorylation Sites in Aged Human Lens Proteins. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1302.

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

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Purpose: : To map the sites of novel thioether modifications on human lens proteins.

Methods: : A human lens membrane fraction was prepared by removing the water-soluble fraction and urea-soluble fraction. The urea-insoluble fraction was either washed with 0.1 M NaOH or not. The remaining pellets were reduced by DTT and alkylated by idoacetamide in 1:1 trifluoethanol (TFE) /50mM ammonium bicarbonate buffer (pH 8.0). The sample was then diluted by 50 mM ammonium bicarbonate (pH 8.0) to reach final TFE of 5% and digested by trypsin. Tryptic peptides were analyzed by Multidimensional Protein Identification Technology (MudPIT) LC-MS/MS methods. The raw data were searched using the TagRecon algorithm in blind modification search mode.

Results: : Modifications to serine and threonine residues with mass addition of 289.073 Da were identified in human lens proteins. The modification was assigned to glutathionylation through a thioether bond based on the accurate mass and tandem mass spectra. Tandem mass spectra of the modified peptides all had a predominant fragment corresponding to loss of a Glu residue from glutathione. The fragmentation pattern was confirmed using a synthetic AQP0 peptide modified by glutathione. This modification was detected on 22 different sites (serine or threonine) in 11 proteins and among these sites, 20 sites have been confirmed as phosphorylation sites. Modified peptides were also detected without NaOH wash which rules out the possibility that this modification forms during NaOH wash. Further analysis of the data yielded modifications by other thiol compounds including a Cys-Gly dipeptide, cysteine and homocysteine.

Conclusions: : Thiol compounds can irreversibly modify serine and threonine residues in aged human lens proteins. Modification most likely occurs at phosphorylation sites through a reaction between thiol compounds and the beta-elimination product, dehydroalanine, that occurs with age. This process likely contributes to the reduced level of glutathione and other antioxidants, the reduced level of phosphorylation, and increased protein-protein crosslinking in the aged lens.

Keywords: protein modifications-post translational • proteomics • aging 

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