title: Global Analysis of Protein Expression of Inner Ear Hair Cells.
abstract: The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.
  
  
  
  Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that hundreds of proteins are uniquely identified or enriched in HCs, extending previous gene expression analyses to reveal novel HC proteins and isoforms. Importantly, deafness-linked proteins were significantly enriched in HCs, suggesting that this in-depth proteomic analysis of IE sensory cells may hold potential for deafness gene discovery.
authors.affiliation: Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.
authors.forename: Ann E
authors.identifier: 0000-0003-1033-9887
authors.initials: AE
authors.lastname: Hickox
authors.affiliation: Departments of Surgery and Neuroscience, University of California, San Diego and Veterans Administration Medical Center, La Jolla, California, 92093.|Translational Neuroscience Facility, Department of Physiology, University of New South Wales, Sydney, New South Wales 2052, Australia, and.
authors.forename: Ann C Y
authors.initials: AC
authors.lastname: Wong
authors.affiliation: Departments of Surgery and Neuroscience, University of California, San Diego and Veterans Administration Medical Center, La Jolla, California, 92093.
authors.forename: Kwang
authors.initials: K
authors.lastname: Pak
authors.affiliation: Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.
authors.forename: Chelsee
authors.initials: C
authors.lastname: Strojny
authors.affiliation: Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.
authors.forename: Miguel
authors.initials: M
authors.lastname: Ramirez
authors.affiliation: Chemical Physiology and Molecular and Cellular Neurobiology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: John R
authors.identifier: 0000-0001-5267-1672
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Departments of Surgery and Neuroscience, University of California, San Diego and Veterans Administration Medical Center, La Jolla, California, 92093, jeffrey.savas@northwestern.edu afryan@ucsd.edu.
authors.forename: Allen F
authors.initials: AF
authors.lastname: Ryan
authors.affiliation: Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, jeffrey.savas@northwestern.edu afryan@ucsd.edu.
authors.forename: Jeffrey N
authors.identifier: 0000-0002-8173-5580
authors.initials: JN
authors.lastname: Savas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002874:Chromosome Mapping; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics* / Q000502:physiology*; D006199:Hair Cells, Auditory, Inner / Q000737:chemistry / Q000378:metabolism*; D006311:Hearing Disorders / Q000235:genetics / Q000473:pathology; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D050821:Transcription Factor Brn-3C / Q000096:biosynthesis / Q000235:genetics; D059467:Transcriptome; D015837:Vestibular Diseases / Q000235:genetics / Q000473:pathology
chemical_list: D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; C121290:Pou4f3 protein, mouse; D050821:Transcription Factor Brn-3C
keywords: cochlea; deafness; hair cells; inner ear; mass spectrometry; proteome