Age-Related Changes in the Spatial Distribution of Human Lens α-Crystallin Products by MALDI Imaging Mass Spectrometry

The ocular lens consists of an epithelial cell monolayer covering the anterior pole, and highly elongated fiber cells, which make up the bulk of the lens cell mass. Throughout life, lens epithelial cells migrate toward the lens equator where they begin to elongate and differentiate into lens fiber cells. Over time, layers of fiber cells are laid down to create a gradient of cell age in which the oldest cells are located in the lens nucleus and the youngest in the lens cortex. During fiber differentiation, potential light-scattering elements such as cell organelles and nuclei are degraded,1,2 which not only removes them from the path of light, but also removes the ability of differentiated lens fiber cells to synthesize new protein. Together with the abundant expression of crystallin proteins in lens fiber cells, which contributes to the gradient of refractive index in the lens,3,4 these vital lens fiber cell–specific adaptations help maintain lens transparency throughout life. Three main classes of crystallin proteins that are present in the mammalian lens—α, β, and γ—together constitute up to 90% of total lens protein.5 A typical mammalian lens contains 30% to 40% α-crystallin,6 and its structure and function have been studied extensively in normal and diseased lens states. A member of the small heat shock protein family, α-crystallin consists of two subunits, αA and αB, which exist as large heterogeneous multimeric complexes with molecular weights ranging from 300,000 to more than 1,000,000.6 Generally, the ratio of αA to αB subunits in the mammalian lens is 3:1. It is well established that α-crystallin acts as both a structural protein and a molecular chaperone binding to denatured lens proteins to prevent nonspecific protein aggregation.7,8 Since there is no protein turnover in differentiated lens fibers, lens proteins, including α-crystallin, undergo many age-related post-translational modifications that include truncation, deamidation, glycation, and phosphorylation, which are thought to induce functional changes.5,9–11 Truncation12–14 and phosphorylation15–18 are known to alter the chaperone activity of α-crystallin. Furthermore, lens protein modifications accumulate over life and may contribute to lens opacification and cataractogenesis by inducing aggregation and water insolubilization of lens proteins in aged and cataractous tissue, although this process is not fully understood.19 Early studies of lens crystallins used whole lens homogenates and one-dimensional gel electrophoresis methodologies to investigate structure and function.20 More recently, micro-dissection techniques producing multiple distinct lens regions,21,22 two-dimensional gel electrophoresis (2-DE),23,24 and mass spectrometric techniques have been used to identify and localize many posttranslational modifications of α-crystallin.25,26 Although immensely powerful, these studies remain limited in the amount of spatial information produced. Recently, MALDI (matrix-assisted laser desorption ionization) imaging mass spectrometry (MALDI tissue imaging) techniques were developed to map the distribution of α-crystallin products in bovine and rabbit lenses.27,28 MALDI tissue imaging is a relatively new proteomic technique that utilizes the molecular specificity of biological mass spectrometry while maintaining spatial information and allows multiple unmodified and modified forms of a protein to be mapped simultaneously, directly from thin tissue sections.29,30 With this technology, multiple truncation products and phosphorylated α-crystallin forms have been detected in the bovine and rabbit lens. In the present study, the MALDI imaging technique has been adapted to study posttranslational processing of human α-crystallin as a function of lens age and disease.