title: Quantitative proteomics analysis by iTRAQ in human nuclear cataracts of different ages and normal lens nuclei.
abstract: OBJECTIVE
  The goal of this study was to quantitatively identify the differentially expressed proteins in nuclear cataracts of different ages and normal lens nuclei in humans.
  
  
  METHODS
  Forty-eight human lens nucleus samples with hardness grades III, IV were obtained during cataract surgery by extracapsular cataract extraction. Seven normal transparent human lens nuclei were obtained from fresh normal cadaver eyes during corneal transplantation surgery. Lens nuclei were divided into seven groups according to age and optic axis: Group A (average age 80.8 ± 1.2 years), Group B (average age 57.0 ± 4.0 years), Group C average age 80.3 ± 4.5 years), Group D (average age 56.9 ± 4.2 years), Group E (average age 78.1 ± 2.5 years), Group F (average age 57.6 ± 3.3 years) and Group G (seven normal transparent human lenses from normal cadaver eyes, average age 34.7 ± 4.2 years). Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were further analyzed using 8-plex iTRAQ labeling combined with 2D-LC-MS/MS. The data were analyzed with the ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated by Western blotting.
  
  
  RESULTS
  We employed biological and technical replicates and selected the intersection of the two results, which included 80 proteins. Nine proteins were differentially expressed among the 80 proteins identified using proteomic techniques. In age-related nuclear cataracts (ARNC), the expression levels of fatty acid-binding protein and pterin-4-alpha-carbinolamine dehydratase were upregulated, whereas the levels of alpha-crystallin B chain (CRYAB), GSH synthetase, phakinin, gamma-crystallin C, phosphoglycerate kinase 1, betaine-homocysteine S-methyltransferase 1 (BHMT1), and spectrin beta chain were downregulated. These proteins may be associated with abnormal protein aggregation and oxidative stress. GSH synthetase and CRYAB expression levels in the nuclear cataract decreased with age. The mass spectrometric analysis results were consistent with the Western blot validation.
  
  
  CONCLUSIONS
  The results indicate that CRYAB and GSH synthetase may be involved in ARNC pathogenesis. iTRAQ combined with 2D-LC-MS/MS provides new methods for future studies of pathological mechanisms and protective drug development for ARNC.
authors.affiliation: Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China.|Department of Ophthalmology, Xi'an Central Hospital, Xi'an, China.
authors.forename: Hai Yan
authors.initials: HY
authors.lastname: Zhou
authors.affiliation: Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Ophthalmology, Xi'an Central Hospital, Xi'an, China.
authors.forename: Li Li
authors.initials: LL
authors.lastname: Wang
authors.affiliation: Department of Clinical Medicine, Xi'an Medical University, Xi'an, China.
authors.forename: Wei Jia
authors.initials: WJ
authors.lastname: Yan
authors.affiliation: Department of Ophthalmology and Visual Sciences, Washington University, Saint Louis, MO, USA.
authors.forename: Ying Bo
authors.initials: YB
authors.lastname: Shui
authors.affiliation: Department of Ophthalmology and Visual Sciences, Washington University, Saint Louis, MO, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Beebe
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000473:pathology*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002386:Cataract / Q000378:metabolism* / Q000473:pathology*; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D005981:Glutathione Synthase / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007907:Lens Nucleus, Crystalline / Q000378:metabolism* / Q000473:pathology; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D038203:alpha-Crystallin B Chain / Q000378:metabolism
chemical_list: C059215:CRYAB protein, human; D005136:Eye Proteins; D010455:Peptides; D038203:alpha-Crystallin B Chain; D005981:Glutathione Synthase
keywords: Alpha-crystallin B chain; GSH synthetase; Human; iTRAQ Nuclear cataract