Abstract
BackgroundVitreous is an accessible, information-rich biofluid that has recently been studied as a source of retinal disease-related proteins and pathways. However, the number of samples required to confidently identify perturbed pathways remains unknown. In order to confidently identify these pathways, power analysis must be performed to determine the number of samples required, and sample preparation and analysis must be rigorously defined.MethodsControl (n = 27) and proliferative diabetic retinopathy (n = 23) vitreous samples were treated as biologically distinct individuals or pooled together and aliquoted into technical replicates. Quantitative mass spectrometry with tandem mass tag labeling was used to identify proteins in individual or pooled control samples to determine technical and biological variability. To determine effect size and perform power analysis, control and proliferative diabetic retinopathy samples were analyzed across four 10-plexes. Pooled samples were used to normalize the data across plexes and generate a single data matrix for downstream analysis.ResultsThe total number of unique proteins identified was 1152 in experiment 1, 989 of which were measured in all samples. In experiment 2, 1191 proteins were identified, 727 of which were measured across all samples in all plexes. Data are available via ProteomeXchange with identifier PXD025986. Spearman correlations of protein abundance estimations revealed minimal technical (0.99–1.00) and biological (0.94–0.98) variability. Each plex contained two unique pooled samples: one for normalizing across each 10-plex, and one to internally validate the normalization algorithm. Spearman correlation of the validation pool following normalization was 0.86–0.90. Principal component analysis revealed stratification of samples by disease and not by plex. Subsequent differential expression and pathway analyses demonstrated significant activation of metabolic pathways and inhibition of neuroprotective pathways in proliferative diabetic retinopathy samples relative to controls.ConclusionsThis study demonstrates a feasible, rigorous, and scalable method that can be applied to future proteomic studies of vitreous and identifies previously unrecognized metabolic pathways that advance understanding of diabetic retinopathy.
Highlights
Vitreous is an accessible, information-rich biofluid that has recently been studied as a source of retinal disease-related proteins and pathways
A similar large-scale approach is needed in ophthalmology, where an aging population and unhealthy lifestyles have led to rapidly growing populations of patients with age-related macular degeneration (AMD) and diabetic retinopathy (DR), vision-threatening diseases which affect more than 100 million patients worldwide [5,6,7]
Potential role of extracellular vesicles In addition to the components of the vitreous proteome discussed above, we have previously shown that human vitreous contains an abundant population of Extracellular vesicle (EV) [14]
Summary
Information-rich biofluid that has recently been studied as a source of retinal disease-related proteins and pathways. A similar large-scale approach is needed in ophthalmology, where an aging population and unhealthy lifestyles have led to rapidly growing populations of patients with age-related macular degeneration (AMD) and diabetic retinopathy (DR), vision-threatening diseases which affect more than 100 million patients worldwide [5,6,7]. Both AMD and DR are heterogeneous in terms of disease onset, progression, and severity and are ill-suited for a one-size-fits-all treatment approach. The absence of usable ocular data continues to restrict such efforts
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