MS/MS in silico subtraction-based proteomic profiling as an approach to facilitate disease gene discovery: application to lens development and cataract
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 high-throughput 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-friendly visualization of promising candidates, thus making iSyTE a comprehensive tool for cataract gene discovery.
The authors thank Drs. Melinda Duncan and Velia Fowler for helpful discussions. This work was supported by National Institutes of Health/National Eye Institute [R01 EY021505 to S.L.]. Support from the University of Delaware Core Imaging Facility and Proteomics and Mass Spectrometry Facility was made possible through the Institutional Development Award (IDeA) from the National Institutes of Health/National Institute of General Medical Sciences INBRE Program Grant [Grant Number P20 GM103446]. Acquisition of the confocal microscope used in this study was funded by the National Institutes of Health/National Center for Research Resources Grant [S10 RR027273]. Mass spectrometric analysis was performed by the OHSU Proteomics Shared Resource with partial support from NIH core Grants P30 EY010572 and P30 CA069533 and shared instrument Grant S10OD-012246. S.A. was supported by a Fight for Sight Summer Student Fellowship and Sigma Xi award.
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Conflict of interest
The authors declare that they have no conflicts of interest.
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