Proteomic analysis of opsins and thyroid hormone-induced retinaldevelopment using isotope-coded affinity tags (ICAT) and massspectrometry

[摘要] Purpose: Analyses that reveal the relative abundance of proteins areinformative in elucidating mechanisms of retinal development and diseaseprogression. However, popular high-throughput proteomic methods do notreliably detect opsin protein abundance, which serve as markers ofphotoreceptor differentiation. We utilized thyroid-hormone (TH)treatment of rainbow trout (Oncorhynchus mykiss) as a model of coneapoptosis and cone regeneration. We used this model to investigate ifemerging proteomic technology allows effective analysis of retinaldevelopment and opsin protein abundance. We also sought to begin acharacterization of proteomic changes in the retina occurring with THtreatment and address whether TH affects proliferation or photoreceptordifferentiation.Methods: Retinal homogenates were prepared from control andTH-treated fish. Peptides from control and treated homogenates weredifferentially labeled, using isotope-code affinity tags (ICAT) andanalyzed using capillary liquid chromatography-electrosprayionization-tandem mass spectrometry (capLC-ESI-MS/MS). This methodidentifies proteins and quantifies their relative abundance between twosamples.Results: The relative abundance of many retinal proteins changedduring TH treatment. These included proteins from every functionalclass. We detected 1,684 different peptides, and our quantificationsuggests that 94 increased and 146 decreased in abundance more than 50%during TH treatment. Cell-cycle proteins appear to be increased,consistent with TH-inducing cell proliferation, similar to its effect inXenopus. Other proteins associated with retinal development, such asΔA and tubulins, changed in abundance during TH treatment. Rodopsin and three cone opsins were identified and the relative abundanceof each changed with TH treatment.Conclusions: ICAT and capLC-ESI-MS/MS are an effective complement toother molecular approaches that investigate the mechanisms of retinaldevelopment. Unlike other proteomic techniques, this approach does notrequire development of species- or tissue-specific methodology, such ascharacterizing two dimensional (2D) gels or antibodies, in order to bepractical as a high-throughput approach. Importantly, this technologywas able to assess the relative abundance of opsin proteins. Thesefindings represent the first high-throughput proteomic analysis of theretina and demonstrate the technique's ability to provide usefulinformation in retinal development.

[学科分类] 生物化学/生物物理