July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Quantitative proteomic analysis of retina in primary and secondary retinal ganglion cell (RGC) degeneration using SWATH-mass spectrometry (MS)
Author Affiliations & Notes
  • Jacky Man Kwong Kwong
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Thomas Chuen Lam
    Optometry, Hong Kong Polytechnic University, Hong Kong
  • F.J. Yu
    Optometry, Hong Kong Polytechnic University, Hong Kong
  • Andes Y.H. Sze
    Optometry, Hong Kong Polytechnic University, Hong Kong
  • King Kit Li
    Optometry, Hong Kong Polytechnic University, Hong Kong
  • Chi-ho To
    Optometry, Hong Kong Polytechnic University, Hong Kong
  • Joseph Caprioli
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Jacky Man Kwong Kwong, None; Thomas Chuen Lam, None; F.J. Yu, None; Andes Y.H. Sze, None; King Kit Li, None; Chi-ho To, None; Joseph Caprioli, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5655. doi:
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      Jacky Man Kwong Kwong, Thomas Chuen Lam, F.J. Yu, Andes Y.H. Sze, King Kit Li, Chi-ho To, Joseph Caprioli; Quantitative proteomic analysis of retina in primary and secondary retinal ganglion cell (RGC) degeneration using SWATH-mass spectrometry (MS). Invest. Ophthalmol. Vis. Sci. 2019;60(9):5655.

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

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Abstract

Purpose : To examine and validate the retinal proteome profile after regional optic nerve damage.

Methods : Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the optic nerve in adult Wistar rats; the contralateral eye was used as a control. The density of RGCs in the retina from 1 to 8 weeks after pONT was topographically quantified with Rbpms antibody. Temporal and nasal retinal samples were collected separately from both eyes at 2 weeks after pONT and soluble proteins (n=4; 3 technical replicates) were subjected to profiling with a high resolution hybrid quadrupole time-of-flight MS running on label-free SWATHTM acquisition (SCIEX). An information dependent acquisition ion library was generated from all individual biological replicates for SWATHTM peptides quantification. MS spectra were searched for protein identification with ProteinPilot 5.0 (SCIEX) using the Paragon algorithm. After multiple linear regression normalization, significantly regulated proteins (P<0.05; FC>1.4 or <0.7) were selected for validation with multiple reaction monitoring (MRM) approach on a QTRAP 6500+ MS (SCIEX).

Results : No significant change in RGC density was noted in the nasal quadrant at 1 week after pONT (n=8) but the percentage loss increased to 43.6±7.7% at 8 weeks (n=15; P=0.0001) while there was 78.9±5.8% and 27.7±6.0% of RGC survival in the temporal quadrant at 1 and 8 weeks respectively. This indicates secondary RGC degeneration in the nasal quadrant, and acute and continued loss of RGCs in the temporal quadrant. A total of 3641 proteins (>25,000 peptides) with FDR=1% were identified in the rat retinas as an ion library. At 2 weeks, 47 and 23 proteins were significantly regulated in temporal and nasal quadrant respectively when compared to control while 13 of them were found in both quadrants. For MRM assay, 11 and 4 regulated proteins from temporal and nasal quadrant respectively were confirmed. MRM data had strong associations with SWATHTM quantification in both temporal (y=1.18x-0.17; R2=0.97) and nasal (y=0.90x-0.06; R2=0.96) quadrants.

Conclusions : Although differential protein expression was observed in both primary and secondary RGC degeneration, more significant changes were found in the site with primary RGC degeneration. This integrated MS approach may help elucidate the proteomic response to localized optic nerve injury.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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