Phosphoproteomics characterization of novel phosphorylated sites of lens proteins from normal and cataractous human eye lenses.
OBJECTIVE
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.
Graduate Institute of Medicine and Center for Research Resources and Development, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chun-Hao
CH
Huang
Yi-Ting
YT
Wang
Chia-Feng
CF
Tsai
Yu-Ju
YJ
Chen
Jiahn-Shing
JS
Lee
Shyh-Horng
SH
Chiou
D000328:Adult; D000368:Aged; D002386:Cataract / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D003459:Crystallins / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry
D003459:Crystallins; D010750:Phosphoproteins; D014443:Tyrosine; D012694:Serine; D014357:Trypsin
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