Proteomic Profiling of Photoreceptor Cells under Simulated Hyperglycemia with SWATH Mass Spectrometry

Diabetic retinopathy (DR) is one of the leading causes of blindness and vision impairment in 2020. Apart from vasculopathy, DR has been found to involve retinal neurons including amacrine cells and retinal ganglion cells. Recent literature also suggested that photoreceptors secret proinflammatory molecules and produce reactive oxygen species that contribute to the development of DR. The effect of hyperglycemia on photoreceptors is not well understood. Here, we aimed to investigate the high glucose-induced changes in the proteomic profile of 661w photoreceptor-like cell line with data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis. 661w cells were incubated in normal glucose (5.5mM glucose) and high glucose (55mM glucose) conditions for 48 hours. The sample (n=3 in each group) were then processed with S-TrapTM Micro Spin Column Digestion (S-Trap) protocol according to the manufacturer’s guideline. The MS data were acquired using a hybrid quadrupole time-offlight TripleTOF 6600 mass spectrometer (SCIEX) with Analyst TF 1.7 software. Data obtained were exported and analyzed using ProteinPilot 5.0.1, PeakView 2.2, MarkerView 1.3 software, and OneOmics cloud-based platform (SCIEX). Pathway analysis was conducted with Ingenuity® Pathway Analysis (IPA). A total of 625 proteins out of 3,613 proteins was found to be differentially expressed (p<0.05, Fold-change ≥ 1.5 or ≤ 0.667) in the 661w cells under high glucose condition compared to normal glucose condition. Three hundred thirteen proteins were found to be up-regulated, while 312 proteins were found to be down-regulated. The top three enriched pathways revealed by IPA included 1) Protein Ubiquitination Pathway, 2) Mitochondrial Dysfunction, and 3) Oxidative Phosphorylation. Our data suggested that high glucose condition may trigger changes in these three metabolic pathways in photoreceptor cells. These changes may have led to compromised mitochondrial function and increased cell apoptosis in the 661w cells previously reported by our team. The alternation of these three pathways has also been shown to affect the physiology of other retinal cell types under simulated hyperglycemia. The underlying molecular changes and their role in the pathogenesis of DR warrant further investigation. This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.