Abstract
Purpose: :
Lens crystallins undergo many post–synthetic modifications including truncation, disulfide bond formation, oxidation, and deamidation. Deamidation of lens crystallins is prevalent in the adult lens and specific deamidation sites have been suggested to be associated with aging and cataracts. Yet these studies have been hindered by the lack of suitable quantitative methods of measurement of protein deamidation without loss of protein. The purpose of this research was to quantitatively measure deamidation of proteins without prior sample preparation or separation in order to directly compare the amidated and deamidated forms.
Methods: :
Donor Eyes were obtained from the Oregon Lions Eye and Hearing Foundation. Lenses were excised and homogenized in 20 mM phosphate buffer (pH 7.0) and 1 mM EDTA with a protease inhibitor cocktail, centrifuged at 20,000 x g and the soluble proteins stored at –70 degrees C. The measurement technique used was ion cyclotron resonance Fourier transform mass spectrometry, FTMS, alone with no prior sample preparation or separation. Recombinantly–expressed human eye lens betaB2–crystallins were used as controls.
Results: :
The betaB2–crystallin mass spectra showed a good quantitative dependence upon extent of deamidation. Direct injection by electrospray ionization followed by ion selection and laser fragmentation or by collision fragmentation produced fragments of amidated and deamidated betaB2–crystallin that were appropriate for FTMS quantitative analysis. Relative amounts of the major lens crystallins were in good agreement with previously published results using two–dimensional electrophoresis and electrospray mass spectrometry.
Conclusions: :
FTMS quantitative analysis of protein deamidation, as reported for the first time herein and illustrated with betaB2–crystallin, should prove quite useful. This procedure omits gel separation, chromatography, enzymatic digestion, derivatization, and other procedures that currently add cost and time while degrading quantitative comparison of the amidated and deamidated forms. The substantial potential significance of this technique is evident, as example, for lens crystallins where it makes possible quantitative studies of age and disease–dependent deamidation that have heretofore been very difficult.
Keywords: proteomics • crystalline lens • protein modifications-post translational