title: Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry.
abstract: Characterization of proteins that mediate mechanotransduction by hair cells, the sensory cells of the inner ear, is hampered by the scarcity of these cells and their sensory organelle, the hair bundle. Mass spectrometry, with its high sensitivity and identification precision, is the ideal method for determining which proteins are present in bundles and what proteins they interact with. We describe here the isolation of mouse hair bundles, as well as preparation of bundle protein samples for mass spectrometry. We also describe protocols for data-dependent (shotgun) and parallel reaction monitoring (targeted) mass spectrometry that allow us to identify and quantify proteins of the hair bundle. These sensitive methods are particularly useful for comparing proteomes of wild-type mice and mice with deafness mutations affecting hair-bundle proteins.
authors.affiliation: Oregon Hearing Research Center, Oregon Health & Science University, Portland, OR, United States; Vollum Institute, Oregon Health & Science University, Portland, OR, United States.
authors.forename: J F
authors.initials: JF
authors.lastname: Krey
authors.affiliation: Oregon Health & Science University, Portland, OR, United States.
authors.forename: P A
authors.initials: PA
authors.lastname: Wilmarth
authors.affiliation: Oregon Health & Science University, Portland, OR, United States.
authors.forename: L L
authors.initials: LL
authors.lastname: David
authors.affiliation: Oregon Hearing Research Center, Oregon Health & Science University, Portland, OR, United States; Vollum Institute, Oregon Health & Science University, Portland, OR, United States; Oregon Health & Science University, Portland, OR, United States. Electronic address: gillespp@ohsu.edu.
authors.forename: P G
authors.initials: PG
authors.lastname: Barr-Gillespie
mesh_terms: D003599:Cytoskeleton / Q000378:metabolism; D006198:Hair Cells, Auditory / Q000378:metabolism; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*
chemical_list: D020543:Proteome
keywords: Actin; Cytoskeleton; Espin; Hair bundles; Hair cells; Ion trap; Orbitrap; Shotgun; Stereocilia; Targeted