June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Isoaspartate in γS-crystallin is associated with protein insolubilization in age-related cataract
Author Affiliations & Notes
  • Larry L David
    Biochemistry & Molecular Biology, Oregon Health Sciences Univ, Portland, Oregon, United States
  • Phillip A Wilmarth
    Biochemistry & Molecular Biology, Oregon Health Sciences Univ, Portland, Oregon, United States
  • Kirsten J Lampi
    Integrative Biosciences, Oregon Health & Science Univ, Portland, Oregon, United States
  • Footnotes
    Commercial Relationships   Larry David, None; Phillip Wilmarth, None; Kirsten Lampi, None
  • Footnotes
    Support  NIH grants R01 EY027012, R01 EY007755, and P30 EY010572
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5594. doi:
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      Larry L David, Phillip A Wilmarth, Kirsten J Lampi; Isoaspartate in γS-crystallin is associated with protein insolubilization in age-related cataract. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5594.

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

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Purpose : To identify isoaspartate sites in aged human lenses and test their association with crystallin insolubilization, a precursor to cataract formation.

Methods : Proteins from the nuclear region of human lenses of ages 82, 75, 88, and 85 years with increasing brunescence were trypsinized, and peptides separated by a 210 min reverse phase chromatography gradient. The elution times of γS peptide isoforms were determined, both with and without deamidation, using a combination of high-resolution survey scans and data-independent MS/MS. Peptides containing putative isoaspartate modifications were then targeted for electron transfer dissociation (ETD) to detect mass shifted c and z fragment ions characteristic of isoaspartate containing peptides.

Results : Targeting peptides for fragmentation using ETD resulted in generation of c +57 ions and z -57 ions upon fragmentation of peptides containing isoasp at either natural D residues, or sites of N deamidation. Assignment of isoasp containing peaks allowed the relative quantification of isoasp (as a total of each deamidated peptide form) for γS peptides containing sites D12, N14, N53, N146, as well as a non-amide peptide containing D161. The extent of single deamidation in peptides containing N14, N53, N146 increased progressively with lens brunescence. In the most brunescent lens, deamidation in the insoluble fraction was 62, 46, and 55% at the three sites; and was 2.4-, 7.5-, and 9.6- fold higher than the percentage in the soluble fraction, respectively. Moreover, the insoluble fraction contained a higher proportion of isoasp containing peptide isoforms than the soluble fraction. The proportion of peptide isoforms containing a combination of both isoasp 12 and isoasp 14 residues were 2.0-fold higher in the insoluble fraction, while isoforms containing isoasp at residues 53 and 143 were in 3.0- and 1.3-fold higher proportions, respectively, than in the soluble fraction. While only 4% of the peptide containing Q170 was deamidated, 21% of D161 in this peptide was converted to isoasp, and the isoasp containing isoforms were 13-fold more abundant in the insoluble fraction.

Conclusions : Isoasp formation in aged lenses can be readily detected by mass spectrometry by targeting peptides for ETD fragmentation. The association of isoasp modifications with crystallin insolubilization suggest that this abundant modification may be a major player in cataract formation.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.


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