Identification of the primary targets of carbamylation in bovine lens proteins by mass spectrometry.
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.
Zhang
Jie
J
Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China.
Yan
Hong
H
Harding
John J
JJ
Liu
Zhen-Xiong
ZX
Wang
Xin
X
Ruan
Yu-Song
YS
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
D038202:alpha-Crystallin A Chain; D038206:beta-Crystallin B Chain; D038222:gamma-Crystallins