Spatiotemporal Mapping of Phosphorylation and Oxidation in the Pig Lens

BackgroundThe developmental pattern of the crystalline lens provides a unique model to study biological aging and its effects on the posttranslational modification of long-lived proteins. The orderly differentiation of lens fiber cells leads to a spatiotemporal gradient where mature, organelle-free fiber cells are packed in the lens nucleus surrounded by con-centric rings of successively younger fiber cells in the cortex. MethodsPig lenses were separated into six layers by dissolution in a hypotonic buffer. The changes in protein abundance, oxidation, and phosphorylation that occur across the spatiotemporal gradient of the lens were assessed quantitatively by using data independent acquisition label-free proteomic analysis of these six fractions. ResultsExpected changes in protein abundance of major lens protein which reflect the maturation process of lens fiber cells across the spatiotemporal gradient were found. Significant differences were noted in phosphorylation sites on crystallins, phakinin, and actin. Significant changes in oxidation of residues on essential lens proteins, as well as several glycolytic enzymes, were found across the spatiotemporal gradient. ConclusionDissolution of the lens followed by high resolution data-independent acquisition proteomics is a powerful technique for spatial mapping of protein abundance and posttranslational modification changes in the lens. The oxidation and phosphorylation sites noted in this study may play important roles in both lens development and cataractogenesis.