title: Identification of the primary targets of carbamylation in bovine lens proteins by mass spectrometry.
abstract: OBJECTIVE
  Carbamylation, an important post-translational modification of proteins, inevitably causes conformational changes of lens proteins. It may increase aggregation between crystallin molecules and disrupt the close packing required for transparency thus leading to cataract. The aim of this study was to isolate the primary targets of carbamylation in the lens and identify them by mass spectrometry.
  
  
  METHODS
  Fresh intact bovine lenses were incubated with 100 mM potassium cyanate for 7 days. The proteins in the water-soluble fractions from the normal control and the cyanate-modified lens proteins were separated by two-dimensional (2-D) gel electrophoresis with identification after silver staining. Protein spots that differed between the normal and carbamylated groups were selected for further analysis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
  
  
  RESULTS
  The 2-D gel results showed that the major lens proteins were in the section of pI 5-8, with relative molecular masses of 20-35 kDa, and changes in the carbamylated fraction like strings of beads indicating modification. The mass spectrometry analysis and a database search identified carbamylated proteins originating from alphaA-crystallin, betaB2- and gammaS-(betaS)-crystallins.
  
  
  CONCLUSIONS
  These crystallins may be vulnerable proteins targeted by carbamylation. The accumulated aggregation and loss of chaperone activity may contribute to cataract formation.
authors.affiliation: Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
authors.forename: John J
authors.initials: JJ
authors.lastname: Harding
authors.forename: Zhen-Xiong
authors.initials: ZX
authors.lastname: Liu
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Yu-Song
authors.initials: YS
authors.lastname: Ruan
mesh_terms: D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000378:metabolism*; D013058:Mass Spectrometry; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000378:metabolism*; D038206:beta-Crystallin B Chain / Q000737:chemistry / Q000378:metabolism*; D038222:gamma-Crystallins / Q000737:chemistry / Q000378:metabolism
chemical_list: D038202:alpha-Crystallin A Chain; D038206:beta-Crystallin B Chain; D038222:gamma-Crystallins