Abstract
Purpose: :
To determine what proteins are found in the human lens and measure changes in their relative abundance and solubility during aging.
Methods: :
A series of human lenses from donors of increasing age (3–day, 18–year, 70–year, and 93–year old) were homogenized and separated into water–soluble (WS) and water–insoluble (WI) proteins by centrifugation. Each fraction was then denatured in urea, reduced/alkylated, digested with trypsin, the complex mixtures of peptides separated by two–dimensional liquid chromatography (2D–LC) and peptide sequences determined by ion trap mass spectrometry. The number of identified tandem mass spectra assigned to each protein was used to estimate changes in the protein's relative abundance and movement into the WI fraction during aging. To allow comparisons between samples, protein spectral counts were normalized by the total count of all identified proteins in that sample. For further comparison, proteins from similar aged lenses were also separated by two–dimensional electrophoresis (2D–E) and abundances measured by densitometry.
Results: :
2D–LC analysis confidently identified 178 different lens proteins, and was far more sensitive than 2D–E in protein detection. Furthermore, many proteins, such as αA–crystallin, were essentially lost from 2D–E gels during aging in the WI fraction, but were still abundant in the WI fraction of aged lenses when analyzed by 2D–LC. A maturationally related decrease in γA, ßA2, ßB3, and increase in γD were observed. Major age–related changes in soluble αA, αB, ßB1, ßB2, ßA3/A1, and ßA4 were not detected. However, ßB2 remained predominately soluble, while γB, C, D, and S were selectively insolubilized. Non–crystallin proteins responded to development and aging differently. Some, such as NDRG1 protein, cofilin–1, beta–2 tubulin, and betaine–homocysteine S–methyltransferase were only abundant in 3–day old lens, while several glycolytic enzymes, sorbitol dehydrogenase, and glutathione synthetase were largely unchanged during aging.
Conclusions: :
2D–LC and spectral counting are useful techniques to globally determine changes in the relative abundance of lens proteins during aging, and are far superior to 2D–E in sensitivity and ability to analyze WI proteins. The methodology should be useful to detect changes in relative protein abundance and solubility during cataract.
Keywords: proteomics • aging • protein structure/function