MS/MS in silico subtraction-based proteomic profiling as an approach to facilitate disease gene discovery: application to lens development and cataract

Abstract

While the bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery) effectively identifies human cataract-associated genes, it is currently based on just transcriptome data, and thus it is necessary to include protein-level information to gain greater confidence in gene prioritization. Here we expand iSyTE through development of a novel proteome-based resource on the lens and demonstrate its utility in cataract gene discovery. We applied highthroughput tandem mass spectrometry (MS/MS) to generate a global protein expression profile of mouse lens at embryonic day (E)14.5, which identified 2371 lens-expressed proteins. A major challenge of high-throughput expression profiling is identification of high-priority candidates among the thousands of expressed proteins. To address this problem, we generated new MS/MS proteome data on mouse whole embryonic body (WB). WB proteome was then used as a reference dataset for performing "in silico WB-subtraction" comparative analysis with the lens proteome, which effectively identified 422 proteins with lens-enriched expression at ≥2.5 average spectral counts, ≥2.0 fold-enrichment (FDR <0.01) cut-off. These top 20% candidates represent a rich pool of high-priority proteins in the lens including known human cataract-linked genes and many new potential regulators of lens development and homeostasis. This rich information is made publicly accessible through iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), which enables user-*

Deficiency of the bZIP transcription factors Mafg and Mafk causes misexpression of genes in distinct pathways and results in lens embryonic developmental defects

“…Validation of key Mafg −/− :Mafk −/− DEGs relevant to lens biology Next, to further prioritize candidate genes relevant to lens biology so as to uncover the underlying molecular changes associated with Mafg −/− :Mafk −/− lens defects, we applied various selection criteria to the list of DEGs identified by RNA-seq. These include gene ontology (GO) and pathway analysis as well as iSyTE analysis, which informs on the expression of candidate genes in normal lens development and its altered expression in various gene perturbation mouse models with lens defects/cataract, and has been shown to be effective in prioritizing key genes and pathways in the lens Frontiers in Cell and Developmental Biology frontiersin.org (Lachke et al, 2011(Lachke et al, , 2012a(Lachke et al, , 2012bWolf et al, 2013;Manthey et al, 2014;Agrawal et al, 2015;Anand et al, 2015Anand et al, , 2018bAnand et al, , 2021Audette et al, 2016;Cavalheiro et al, 2017;Patel et al, 2017;Kakrana et al, 2018;Krall et al, 2018;Siddam et al, 2018;Padula et al, 2019;Aryal et al, 2020;Barnum et al, 2020;Choquet et al, 2021). We also considered the potential relevance of the candidate genes to lens biology based on their function described in the published literature.…”

A Novel Mutation in Cse1l Disrupts Brain and Eye Development with Specific Effects on Pax6 Expression

“…This neural crest expression is also consistent with many of the phenotypes we observe in anteater mutants ( Figure 1 ). iSyTE analysis [ 25 , 26 , 27 ] shows robust Cse1l expression in the mouse lens during development and at adult stages ( Figure S2 ). By E14.5, discrete regions of Cse1l expression were detected in the tooth buds, salivary glands, and tongue (data not shown).…”

Modeling ocular lens disease in Xenopus

“…Understanding the involvement of these genes in cataract can be achieved by constructing and characterizing new animal models inactivated for these genes. Indeed, the bioinformatics resource tool iSyTE has predicted several promising candidates that need to be validated in animal models . The cost‐effective pipeline of cataract investigation in Xenopus that we describe here would help to uncover the genetic basis of these rare genetic cataracts as well as to identify/validate new cataract‐linked genes.…”