title: Identification of in vivo phosphorylation sites of lens proteins from porcine eye lenses by a gel-free phosphoproteomics approach.
abstract: OBJECTIVE
  Phosphorylation is an important post-translational modification for the cellular regulation of various biosignaling pathways. We have identified in vivo phosphorylation sites of various lens proteins including especially the major structural proteins of the crystallin family from porcine eye lenses by means of two-dimensional gel electrophoresis (2-DE) or immobilized metal affinity chromatography (IMAC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
  
  
  METHODS
  For the identification of phosphorylated residues in various lens proteins of porcine lens extracts, we have adapted two complementary proteomic approaches, i.e., pre-fractionation of protein samples with 2-DE or enrichment of phosphopeptides with IMAC followed by LC-MS/MS analysis and database search. The results were compared and validated with those in phosphoproteomics databases.
  
  
  RESULTS
  Two subunits of alpha-crystallin, alphaA-crystallin and alphaB-crystallin, as well as other lens crystallins and non-crystallin cellular proteins, such as beta-enolase, heat shock protein beta-1 (HSP27), and glucose-6-phosphate isomerase (GPI) were found to be phosphorylated in vivo at specific sites. Moreover, alphaA- and alphaB-crystallins were found to be the most abundantly phosphorylated proteins in porcine lenses, being extensively phosphorylated on serine or threonine, but not on tyrosine residues.
  
  
  CONCLUSIONS
  The complementary gel-based and gel-free proteomic strategies have been compared and evaluated for the study of crystallin phosphorylation from whole tissue extracts of porcine eye lenses. Technically, the IMAC method facilitates direct site-specific identification of phosphorylation residues in lens proteins, which does not necessitate the pre-MS/MS 2-DE separation of protein samples. Moreover, the improved strategy using gel-free phosphoproteomics analysis affords a more effective and simplistic method for the determination of in vivo phosphorylation sites than the conventional 2-DE pre-separation of protein mixture. This study should form a firm basis for the comprehensive analysis of post-translational modification of lens proteins in terms of aging or various diseased states.
authors.affiliation: Graduate Institute of Medicine and Center for Research Resources and Development, Kaohsiung Medical University, Kaohsiung, Taiwan. shchiou@kmu.edu.tw
authors.forename: Shyh-Horng
authors.initials: SH
authors.lastname: Chiou
authors.forename: Chun-Hao
authors.initials: CH
authors.lastname: Huang
authors.forename: I-Liang
authors.initials: IL
authors.lastname: Lee
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Wang
authors.forename: Nai-Yu
authors.initials: NY
authors.lastname: Liu
authors.forename: Yeou-Guang
authors.initials: YG
authors.lastname: Tsay
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D034421:Sus scrofa; D053719:Tandem Mass Spectrometry; D014020:Tissue Extracts; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000378:metabolism
chemical_list: D003459:Crystallins; D055551:HSP27 Heat-Shock Proteins; D010750:Phosphoproteins; D020543:Proteome; D014020:Tissue Extracts; D038203:alpha-Crystallin B Chain; D010751:Phosphopyruvate Hydratase; D005956:Glucose-6-Phosphate Isomerase