June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Investigating the Proteome of the ocular lens at multiple developmental time points through mass spectrometry-based protein sequencing
Author Affiliations & Notes
  • Shahid Yar Khan
    The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Muhammad Ali
    The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Chan-Hyun Na
    McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Akhilesh Pandey
    McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Sean Hackett
    The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • S Amer Riazuddin
    The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
    McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Shahid Khan, None; Muhammad Ali, None; Chan-Hyun Na, None; Akhilesh Pandey, None; Sean Hackett, None; S Amer Riazuddin, None
  • Footnotes
    Support  R01EY022714
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1719. doi:
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      Shahid Yar Khan, Muhammad Ali, Chan-Hyun Na, Akhilesh Pandey, Sean Hackett, S Amer Riazuddin; Investigating the Proteome of the ocular lens at multiple developmental time points through mass spectrometry-based protein sequencing. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1719.

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

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Abstract

Purpose : The proteome is the entire repertoire of proteins present in a cell at any time. We undertook mass spectrometry-based proteome sequencing to gain insight into the developing mouse lens and to establish a resource for characterizing the critical components of lens development.

Methods : We extracted mouse lenses at six developmental time points, including two embryonic (E15 and E18) and four postnatal time points (P0, P3, P6, and P9). The ocular tissue was maintained in three distinct pools to serve as biological replicates for each time point. Total protein was isolated, digested with trypsin and labeled with isobaric tandem mass tags (TMT). Three independent 6-plex TMT experiments were performed. The digested, labeled peptides were pooled and fractionated into 96 samples by basic pH reversed-phase liquid chromatography, followed by concatenation into 24 fractions. Tandem mass spectra were collected with an Orbitrap Fusion Lumos mass spectrometry instrument using Synchronous Precursor Selection MS3. The raw data from MS was analyzed with Proteome Discoverer 2.1 and differential expression was performed with Perseus software.

Results : A total of 6,712 proteins were identified (in at least one of the three TMT sets) in the mouse lens at the above-mentioned six developmental time points. Of these, 6,675 proteins were reliably quantitated, and 5,029 proteins were identified in at least two of the TMT sets. Crystallin and heat shock proteins constituted the most abundantly expressed species in the lens proteome. A total of 98 proteins exhibited differential expression (up- or down-regulated) during the developmental time course compared to their levels at embryonic day 15. We identified a total of 321 autophagy-associated proteins in the lens proteome. Comparative analysis of the proteome data with our previously published lens transcriptome identified 5,606 common entries present both in the proteome and transcriptome data sets.

Conclusions : We have established a comprehensive profile of the developing murine lens proteome through mass spectrometry-based proteome sequencing. To the best of our knowledge, this is the first report investigating a comprehensive lens proteome at six developmental time points. This repository will be fundamental in elucidating the processes essential for the development of the ocular lens and the maintenance of its transparency.

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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