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J Proteome Res. 2015 May 1;14(5):2109-2120. doi: 10.1021/pr501238m. Epub 2015 Apr 6.

Proteomic profiling of the retinas in a neonatal rat model of oxygen-induced retinopathy with a reproducible ion-current-based MS1 approach.

Tu C#1,2, Beharry KD#3,4,5, Shen X1,2, Li J1,2, Wang L6, Aranda JV3,4,5, Qu J1,2.

Author information

1
Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14260, United States.
2
New York State Center of Excellence in Bioinformatics and Life Sciences, 701 Ellicott Street, Buffalo, New York 14203, United States.
3
Department of Pediatrics, Division of Neonatal-Perinatal Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, United States.
4
Department of Ophthalmology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, United States.
5
SUNY Eye Institute, Syracuse, New York 13202, United States.
6
The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China.
#
Contributed equally

Abstract

Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.

KEYWORDS:

label-free quantification; neovascularization; oxygen-induced retinopathy; peptide ion current areas; retinopathy of prematurity

PMID:
25780855
PMCID:
PMC4456185
DOI:
10.1021/pr501238m
[Indexed for MEDLINE]
Free PMC Article

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