July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Imaging Mass Spectrometry of Age-Related Molecular Changes in the Ocular Lens
Author Affiliations & Notes
  • Kevin L Schey
    Biochemistry, Vanderbilt University, Nashville, Tennessee, United States
    The Binding Site, Inc., Rochester, Minnesota, United States
  • David MG Anderson
    Biochemistry, Vanderbilt University, Nashville, Tennessee, United States
  • Dhananjay Sakrikar
    The Binding Site, Inc., Rochester, Minnesota, United States
  • Jamie L Wenke
    Biochemistry, Vanderbilt University, Nashville, Tennessee, United States
  • Md. Amir Hossen
    Biochemistry, Vanderbilt University, Nashville, Tennessee, United States
  • Jeffrey M Spraggins
    Biochemistry, Vanderbilt University, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Kevin Schey, None; David Anderson, None; Dhananjay Sakrikar, None; Jamie Wenke, None; Md. Hossen, None; Jeffrey Spraggins, None
  • Footnotes
    Support  NIH Grant EY013462, NIH Grant EY024258
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3483. doi:
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    • Get Citation

      Kevin L Schey, David MG Anderson, Dhananjay Sakrikar, Jamie L Wenke, Md. Amir Hossen, Jeffrey M Spraggins; Imaging Mass Spectrometry of Age-Related Molecular Changes in the Ocular Lens. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3483.

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

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Abstract

Purpose : Many age-related modifications to lens proteins have been discovered through analysis of lens homogenates. The purpose of this study is to use the technology of imaging mass spectrometry (IMS) to spatially localize age-related changes in lens proteins and lipids.

Methods : Frozen sections were cut to 10-12 micron thickness from lenses of varying age and placed on MALDI target plates. MALDI matrices, 1,5-diaminonaphthalene for lipids, sinapinic acid for proteins, or alpha-cyano-4-hydroxycinnamic acid for peptides, were applied by robotic sprayer (proteins & peptides), robotic spotter (membrane proteins), or by sublimation (lipids). Imaging mass spectrometry data were acquired by MALDI-TOF or FTICR instrumentation with spatial resolutions between 10 and 100 microns. On-tissue digestion was accomplished by spraying trypsin solution onto the tissue and, after incubation in a humidity chamber, MALDI matrix was applied. Images of low abundance signals were enhanced in FTICR imaging experiments using continuous accumulation of selected ions (CASI) to enhance specific mass regions. Imaging data were processed and analyzed using Bruker FlexImaging software. Identification of specified analytes was accomplished by tissue extraction followed by LC-MS/MS analysis.

Results : Age-related molecular changes were observed in specific lens regions for soluble lens proteins, membrane proteins, and lipids. Intact unmodified protein signals were observed only in the outer cortical region of all lenses examined. Specific protein modifications imaged include truncation, deamidation, phosphorylation, and glutathionylation. The most abundant modifications were observed for crystallins and AQP0. On-tissue tryptic digestion allowed for large, insoluble, and modified proteins to be imaged by their tryptic peptides. Such proteins include aquaporin-0, MP20, filensin, phakinin, and deamidated crystallins. Spatially defined rings of modified peptides and proteins suggest an age-dependency on such modifications. In aged lenses, highly abundance crystallin peptides as well as cataract-specific peptides were detected in the lens nucleus. Changes in lipid populations with fiber cell age were also observed.

Conclusions : IMS technology provides spatially-resolved analysis of age-related and cataract-specific modifications to lens proteins and lipids.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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