Quantitative measurement of young human eye lens crystallins by direct injection Fourier transform ion cyclotron resonance mass spectrometry

Abstract

Purpose: Human eye lenses at birth are primarily constructed of 12 distinct crystallins and two truncated crystallins. The molecular weights of these 14 proteins vary between about 20,000 and 30,000 Da. The relative amounts of these molecules and their post-synthetic changes with age are of substantial interest in the study of lens biochemistry and lens pathology. Fourier transform mass spectrometry of unfractionated lens homogenates now permits precise quantitative measurement of the relative amounts of lens crystallins. We report herein the measurement of the 14 crystallins in 10 pairs of lenses from humans between the ages of 2 and 300 days.

Methods: Eye lenses were obtained from human donors of various ages in the first year of life. These lenses were homogenized in 0.02 M phosphate buffer at pH 7.0 with 0.001 M EDTA, desalted by washing over a 3,000 Da filter, and injected directly into the nanospray source of a hybrid Fourier transform ion cyclotron resonance mass spectrometer, Qq-FT(ICR)MS, equipped with a 12 Tesla magnet. The crystallins were quantitatively ionized and mass analyzed in the ICR cell of the mass spectrometer. The detected signals of all of the isotopic and charge state species for each crystallin were normalized and summed to determine the protein quantities.

Results: The relative amounts of the 14 crystallins are found to be quite similar from individual to individual at birth. These amounts are in integer ratios to one another that suggest important structural relations within the lens. In two cases, the relative amounts of alphaA- and betaB2-crystallin change proportionally to the logarithm of age during the first year, with alphaA- decreasing and betaB2-crystallin increasing. The changes in alphaA- and betaB2-crystallin are mutually offsetting, with alphaA-crystallin decreasing from 30% to 18% and betaB2-increasing from 12% to 24%.

Conclusions: These observations suggest that the human eye lens at birth is constructed of crystallins in which the numbers of crystallin molecules have regular integral relationships to each other. As the lens develops during the first year, some of these relationships change. While the functional significance of the reciprocal decrease in alphaA- and increase in betaB2-crystallin is not known, betaB2-crystallin may substitute for alphaA-crystallin in the lens structures synthesized during the year after birth. Direct injection FT(ICR)MS of unfractionated lens was found to be an excellent method for the quantitative measurement of lens crystallins.