Microscale analysis of proteins in inner ear tissues and fluids with emphasis on endolymphatic sac, otoconia, and organ of Corti

Here we describe preparatory techniques adapted for the study of proteins of inner ear tissues and fluids that have allowed us to apply state-of-the-art analytical techniques in spite of the minute size and anatomical complexities of this organ. Illustrative examples address unresolved issues of functional and clinical significance. First, we demonstrate how quick-freezing and freeze drying prevents artifacts that arise from sampling endolymphatic sac (ES) content in the liquid state. This set the stage for the generation of the first protein profile of the ES. Identification of crucial proteins will help elucidate mechanisms of endolymph volume regulation and pathogenesis of Meniere's disease. Second, we show how a unique situation allowed identification of otoconial proteins by mass spectrometric analysis without prior separation and we discuss possible roles for these minor otoconins in otoconial development and prevention of degenerative diseases that affect balance. Finally, we demonstrate techniques for the precise dissection of organ of Corti and its substructures, while preserving their near normal chemical state. We extended an earlier study in which we identified a novel calcium-binding protein by IEF, oncomodulin, localized in the outer hair cells and show here the applicability of prefractionation for the screening of calcium-binding proteins of organ of Corti. These studies demonstrate how advanced preparatory and analytical techniques can be applied to studies of the inner ear.