Quantitative measurement of young human eye lens crystallins bydirect injection Fourier transform ion cyclotron resonance massspectrometry

[摘要] Purpose: Human eye lenses at birth are primarily constructed of 12distinct crystallins and two truncated crystallins. The molecularweights of these 14 proteins vary between about 20,000 and 30,000 Da.The relative amounts of these molecules and their post-synthetic changeswith age are of substantial interest in the study of lens biochemistryand lens pathology. Fourier transform mass spectrometry ofunfractionated lens homogenates now permits precise quantitativemeasurement of the relative amounts of lens crystallins. We reportherein the measurement of the 14 crystallins in 10 pairs of lenses fromhumans between the ages of 2 and 300 days.Methods: Eye lenses were obtained from human donors of various agesin the first year of life. These lenses were homogenized in 0.02 Mphosphate buffer at pH 7.0 with 0.001 M EDTA, desalted by washing over a3,000 Da filter, and injected directly into the nanospray source of ahybrid Fourier transform ion cyclotron resonance mass spectrometer,Qq-FT(ICR)MS, equipped with a 12 Tesla magnet. The crystallins werequantitatively ionized and mass analyzed in the ICR cell of the massspectrometer. The detected signals of all of the isotopic and chargestate species for each crystallin were normalized and summed todetermine the protein quantities.Results: The relative amounts of the 14 crystallins are found to bequite similar from individual to individual at birth. These amounts arein integer ratios to one another that suggest important structuralrelations within the lens. In two cases, the relative amounts of αA- and βB2-crystallin change proportionally to the logarithm of ageduring the first year, with αA- decreasing and βB2-crystallinincreasing. The changes in αA- and βB2-crystallin aremutually offsetting, with αA-crystallin decreasing from 30% to 18%and βB2-increasing from 12% to 24%.Conclusions: These observations suggest that the human eye lens atbirth is constructed of crystallins in which the numbers of crystallinmolecules have regular integral relationships to each other. As the lensdevelops during the first year, some of these relationships change.While the functional significance of the reciprocal decrease in αA- and increase in βB2-crystallin is not known, βB2-crystallinmay substitute for αA-crystallin in the lens structuressynthesized during the year after birth. Direct injection FT(ICR)MS ofunfractionated lens was found to be an excellent method for thequantitative measurement of lens crystallins.

[学科分类] 生物化学/生物物理