A Validated Analysis Pipeline for Mass Spectrometry-Based Vitreous Proteomics: Insights Into Proliferative Diabetic Retinopathy
Abstract Background: Vitreous 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.Methods: Control (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 10plexes. Pooled samples were used to normalize the data across plexes and generate a single data matrix for downstream analysis. Results: The total number of unique proteins identified was 1,152 in experiment 1, 989 of which were measured in all samples. In experiment 2, 1,191 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 10plex, 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.Conclusions: This study demonstrates a feasible, rigorous, and scalable method that can be applied to future proteomic studies of vitreous sampled directly from patients in order to advance understanding of pathways altered in retinal disease.