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Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses

PURPOSE: Post-translational modification (PTM) of lens proteins is believed to play various roles in age-related lens function and development. Among the different types of PTM, phosphorylation is most noteworthy to play a major role in the regulation of various biosignaling pathways in relation to...

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Autores principales: Huang, Chun-Hao, Wang, Yi-Ting, Tsai, Chia-Feng, Chen, Yu-Ju, Lee, Jiahn-Shing, Chiou, Shyh-Horng
Formato: Texto
Lenguaje:English
Publicado: Molecular Vision 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025096/
https://www.ncbi.nlm.nih.gov/pubmed/21264232
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author Huang, Chun-Hao
Wang, Yi-Ting
Tsai, Chia-Feng
Chen, Yu-Ju
Lee, Jiahn-Shing
Chiou, Shyh-Horng
author_facet Huang, Chun-Hao
Wang, Yi-Ting
Tsai, Chia-Feng
Chen, Yu-Ju
Lee, Jiahn-Shing
Chiou, Shyh-Horng
author_sort Huang, Chun-Hao
collection PubMed
description PURPOSE: Post-translational modification (PTM) of lens proteins is believed to play various roles in age-related lens function and development. Among the different types of PTM, phosphorylation is most noteworthy to play a major role in the regulation of various biosignaling pathways in relation to metabolic processes and cellular functions. The present study reported the quantitative analysis of the in vivo phosphoproteomics profiles of human normal and cataractous lenses with the aim of identifying specific phosphorylation sites which may provide insights into the physiologic significance of phosphorylation in relation to cataract formation. METHODS: To improve detection sensitivity of low abundant proteins, we first adopted SDS-gel electrophoresis fractionation of lens extracts to identify and compare the protein compositions between normal and cataractous lenses, followed by tryptic digestion, enrichment of phosphopeptides by immobilized metal affinity chromatography (IMAC) and nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS) analysis. RESULTS: By comprehensively screening of the phosphoproteome in normal and cataractous lenses, we identified 32 phosphoproteins and 73 phosphorylated sites. The most abundantly phosphorylated proteins are two subunits of β-crystallin, i.e., βB1-crystallin (12%) and βB2-crystallin (12%). Moreover, serine was found to be the most abundantly phosphorylated residue (72%) in comparison with threonine (24%) and tyrosine (4%) in the lens phosphoproteome. The quantitative analysis revealed significant and distinct changes of 19 phosphoproteins corresponding to 28 phosphorylated sites between these two types of human lenses, including 20 newly discovered novel phosphorylation sites on lens proteins. CONCLUSIONS: The shotgun phosphoproteomics approach to characterize protein phosphorylation may be adapted and extended to the comprehensive analysis of other types of post-translational modification of lens proteins in vivo. The identification of these novel phosphorylation sites in lens proteins that showed differential expression in the cataractous lens may bear some unknown physiologic significance and provide insights into phosphorylation-related human eye diseases, which warrant further investigation in the future.
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spelling pubmed-30250962011-01-24 Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses Huang, Chun-Hao Wang, Yi-Ting Tsai, Chia-Feng Chen, Yu-Ju Lee, Jiahn-Shing Chiou, Shyh-Horng Mol Vis Research Article PURPOSE: Post-translational modification (PTM) of lens proteins is believed to play various roles in age-related lens function and development. Among the different types of PTM, phosphorylation is most noteworthy to play a major role in the regulation of various biosignaling pathways in relation to metabolic processes and cellular functions. The present study reported the quantitative analysis of the in vivo phosphoproteomics profiles of human normal and cataractous lenses with the aim of identifying specific phosphorylation sites which may provide insights into the physiologic significance of phosphorylation in relation to cataract formation. METHODS: To improve detection sensitivity of low abundant proteins, we first adopted SDS-gel electrophoresis fractionation of lens extracts to identify and compare the protein compositions between normal and cataractous lenses, followed by tryptic digestion, enrichment of phosphopeptides by immobilized metal affinity chromatography (IMAC) and nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS) analysis. RESULTS: By comprehensively screening of the phosphoproteome in normal and cataractous lenses, we identified 32 phosphoproteins and 73 phosphorylated sites. The most abundantly phosphorylated proteins are two subunits of β-crystallin, i.e., βB1-crystallin (12%) and βB2-crystallin (12%). Moreover, serine was found to be the most abundantly phosphorylated residue (72%) in comparison with threonine (24%) and tyrosine (4%) in the lens phosphoproteome. The quantitative analysis revealed significant and distinct changes of 19 phosphoproteins corresponding to 28 phosphorylated sites between these two types of human lenses, including 20 newly discovered novel phosphorylation sites on lens proteins. CONCLUSIONS: The shotgun phosphoproteomics approach to characterize protein phosphorylation may be adapted and extended to the comprehensive analysis of other types of post-translational modification of lens proteins in vivo. The identification of these novel phosphorylation sites in lens proteins that showed differential expression in the cataractous lens may bear some unknown physiologic significance and provide insights into phosphorylation-related human eye diseases, which warrant further investigation in the future. Molecular Vision 2011-01-19 /pmc/articles/PMC3025096/ /pubmed/21264232 Text en Copyright © 2011 Molecular Vision. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Huang, Chun-Hao
Wang, Yi-Ting
Tsai, Chia-Feng
Chen, Yu-Ju
Lee, Jiahn-Shing
Chiou, Shyh-Horng
Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses
title Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses
title_full Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses
title_fullStr Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses
title_full_unstemmed Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses
title_short Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses
title_sort phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025096/
https://www.ncbi.nlm.nih.gov/pubmed/21264232
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