A comprehensive description of the identities, concentrations, and interactions of proteins found in hair bundles, the mechanically sensitive organelles of the inner ear, will allow a thorough molecular description of mechanotransduction and hair-bundle assembly. Mass spectrometry methods are now sufficiently sensitive to apply to scarce hair bundles; indeed, our preliminary experiments have identified 56 proteins from a preparation of purified chicken bundles. In this project, we will (1) identify those proteins that are enriched in hair bundles using iTRAQ differential labeling and liquid-chromatography-tandem mass spectrometry (LC-MS/MS), (2) determine the concentrations of key bundle proteins using multiple reaction monitoring techniques and LC-MS/MS, and (3) measure the concentrations of critical bundle proteins during development using both sets of methods. These three aims will serve as pilot experiments to allow systematic application of tandem mass spectrometry to biochemical characterization of hair-bundle function. This project will allow development of sufficiently sensitive mass spectrometry methods to achieve our eventual goal of routinely measuring the concentrations and modification states of hundreds of hair bundle proteins in a single LC-MS/MS experiment.
| Shin, Jung-Bum; Krey, Jocelyn F; Hassan, Ahmed et al. (2013) Molecular architecture of the chick vestibular hair bundle. Nat Neurosci 16:365-74 |
| Zhao, Hongyu; Williams, Diane E; Shin, Jung-Bum et al. (2012) Large membrane domains in hair bundles specify spatially constricted radixin activation. J Neurosci 32:4600-9 |
| Shin, Jung-Bum; Longo-Guess, Chantal M; Gagnon, Leona H et al. (2010) The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice. J Neurosci 30:9683-94 |
| Shin, Jung-Bum; Gillespie, Peter G (2009) Unraveling cadherin 23's role in development and mechanotransduction. Proc Natl Acad Sci U S A 106:4959-60 |
