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KO3-4_5-10ng_75um_140min_20120209_VE2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/PPG1121_PLSWT3-4_5-10ng_75um_140min_20120210_VE2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/PPG1121_PLSWT2-1_5-10ng_75um_140min_20120207_VE2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/PPG1121_PLSKO1-6_5-10ng_75um_140min_20120202_VE2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/PPG1107_PLS-4_5-10ng_75um_140min_20120112_VE2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/SEARCH.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/PEAK.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/RESULT.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/09/PXD004044/OTHER.zipprimaryOK200003690gillespp@ohsu.eduPeter Barr-GillespieMass SpectrometryShotgun proteomicsNot availableStereociliaFimbrinPlastinEspinHair cellsUtricleFascinVestibularhttp://www.ebi.ac.uk/pride/archive/projects/PXD004044UtricleInner EarVestibular Hair CellUtricles were dissected from wild-type or Pls1-null mice; hair bundles were isolated using the twist-off technique. Proteins were digested in-gel with trypsin, and peptides were analyzed by LC-MS/MS using an LTQ-Velos ion-trap mass spectrometer.PrideNot availableiodoacetamide derivatized residueSEQUEST (v.28, rev. 12) was used to search MS2 spectra against version 62 of the Ensembl mouse protein database, with concatenated sequence-reversed entries to estimate error thresholds, 179 common contaminant sequences and their reversed forms, and an entry corresponding to the short form of XIRP2, an abundant hair bundle protein). Database processing was performed using custom Python scripts (http://www.ProteomicAnalysisWorkbench.com). SEQUEST searches for all samples were performed with trypsin enzyme specificity. Average parent ion mass tolerance was 2.5 Da. Monoisotopic fragment ion mass tolerance was 1.0 Da. A variable modification of +16.0 Da on methionine residues was also allowed. Peptides identified with SEQUEST were assembled into proteins the PAW pipeline. Proteins were quantified using MS2 intensities, normalized for molecular mass. To test whether any proteins were differentially expressed in wild-type and Pls1-/- mice, we used an empirical Bayes test with adjustment for false-discovery due to multiple comparisons. Proteins were not filtered by number of identification per condition, but rather as many proteins as possible were kept as long as the model could be fit. The computation was done using the limma package in R Statistical Computing Environment (www.r-project.org)ProteomicsPeter Barr-GillespieLTQ VelosPARTIALOregon Hearing Research Center Oregon Health & Science University Portland OR 97239 USAMus Musculus (mouse)Not availablegillespp@ohsu.eduBiologicalOregon Hearing Research CenterUnited Statesliquid chromatography tandem mass spectroscopy, pdt, 3.4.22.-, setulae, SCS, ion, TRAP, p50, Laboratory, Ska10, Xtwist, Ly62, Mus domesticus, TAPK, A4, Gene, mini-ICE, CASP-14, TR-AP, House Mouse, TYPE, RP11-508D10.1, LC-MS-MS, Hair, gp39., DAGA4, peptide, Polypeptides, Tnfsf5, peptido, AI427122, House, LC-MSMS, GP39, Gene Products, Mus musculus domesticus, AA960487, CD40-L, MAM, SCG3, Mice, Lanugo, CD154, T-BAM, type 5 acid phosphatase, Fetal Hairs, LCMSMS, tartrate-resistant acid ATPase, Fetal Hair, peptides, GKLP, CATC4, Swiss, coat/ hair, iones, vibrissa, beta-Trypsin, Swiss Mice, proteins, twist1, ions, twist2, PCTAIRE2-binding protein, TRAP3, T-cell antigen Gp39, M-Twist, Tudor repeat associator with PCTAIRE-2, MGC:45012, TEIF, quill, Fetal, Trap, HIGM1, peptidos, bHLHa38, TNFSF5, T5ap, MGC - 45012, C78062, LC-MS2, CD40L, fur, P105, NTKL, PCTAIRE2BP, Tnfrsf5, Proteins, mouse, LC-MS/MS, Cd40l, Hairs, Bp50, ACP5, SPENCDI, Peptide, LGMD2C, hCD40L, polypeptide, LC/MS/MS, Ionen, Mus, Mini-ICE, Protein, IMD3, TNF-related activation protein, Ska<m10Jus>, Ly-62, twist, X-twi, Mus musculus, CRS, whisker, Xtwi, DMDA1, Caspase-14 subunit p10, mice, TWIST, Pde, Twist, Swiss Mouse, NKTL, hair, Tripcellim, AW743063, House Mice, Caspase-14 subunit p19, AI326936, MICE, beta Trypsin, microchaeta, CSO, Laboratory Mice, domesticus, Protein Gene Products, Gene Proteins, Trypure, DMDA, Ska-lt-m10Jus-gt-, Ion, liquid chromatography-tandem mass spectroscopy, TrATPase, SCARMD2, IGM, BPES3, BPES2, CRS1, liquid chromatography tandem mass spectrometry, Peptid, CD40LG, Mouse, Polypeptide, TRACP, coat hair, Laboratory Mouse, ACS3Forms, setulae, distal segment of free limb, Laboratory, Mus domesticus, autopodial limb segment, A4, CASP-14, protein, NK/GPI., House Mouse, Hair, peptide, Polypeptides, protein polypeptide chains, L-Isomer Methionine, Drug Tolerance, Methionine, peptido, diseases, Impacts, diseases and disorders, Environmental Impacts, protein aggregate, Gpi-1, WMS, average, pod, human disease, Fetal Hairs, F, Dlp1, peptides, Nlk, M, long, vibrissa, E430039A18Rik, Swiss Mice, proteins, number of, foot, hand, Environmental Impact, Fetal, manus/pes, Homo sapiens disease, house mouse, GPHYSD2, Racemethionine, drug tolerance, SGS, Gpi, immune system tolerance, mouse, 2-Amino-4-(methylthio)butyric acid, Hairs, extra or missing physical or functional parts, Normalcy, ACMICD, shortened, paw, paw/hand/foot/hoof, mOC-X, autopodium, Mini-ICE, Immunologic Tolerance, Methionin, methionine, NK|GPI, Hmet, parent ion, Mus musculus, ORG, Org, whisker, Caspase-14 subunit p10, MS/MS, mice, Drp1, autopodial element, Swiss Mouse, common, Gpi1-r, Gpi1-s, Caspase-14 subunit p19, MASS, MICE, Gpi1-t, beta Trypsin, domesticus, disease, Health, DMDA, Self Tolerance, precursor ion, Tolerance, Mouse, Polypeptide, short, Liquimeth, Bglap-rs1, Gpi1s, AI450666, Dnmlp1, 3.4.22.-, other disease, 2-amino-4-(methylsulfanyl)butanoic acid, False, AI461847, number, L Isomer, FBN, Gene, mini-ICE, precursor, protein-containing complex, TYPE, DAGA4, python, polypeptide chain, AI427122, House, ECTOL1, Gene Products, L-Methionine, Mus musculus domesticus, disease or disorder, Pgi, MAM, SCG3, Mice, Pedameth, Lanugo, Normalities, Fetal Hair, autopodium region, autopod, Swiss, Gpi-1r, MS2, coat/ hair, Gpi-1s, Phi, beta-Trypsin, Gpi-1t, stubby, CD156, OCTD, non-neoplastic, 2-amino-4-(methylthio)butanoic acid, quill, has or lacks parts of type, disorder, peptidos, metionina, fur, DL-Methionine, protein complex, Proteins, disorders, medical condition, backward, MF, L-Isomer, Amf, Peptide, LGMD2C, Impact, mereological quality, polypeptide, Environmental, native protein, Immune Tolerance, natural protein, Mus, Protein, sequence, condition, MFS1, tandem MS, Data Base, WMS2, Met, NK, DMDA1, Normality, distal free limb segment, hair, Tripcellim, House Mice, CD156a, 6330417M19Rik, autopodial segment, primary structure of sequence macromolecule, microchaeta, Laboratory Mice, Protein Gene Products, Trypure, Gene Proteins, SSKS, SCARMD2, cardinality, alpha-amino-gamma-methylmercaptobutyric acid, Environments, Peptid, coat hair, Laboratory Mouse, distal segment of limb, reversed, Immunological Tolerancesetulae, DmelCG5178, I-fimbrin protein, act 87E, DmelCG7478, PLS3 protein, Laboratory, Mus domesticus, Stereocilium, A4, CASP-14, protein, Act5, House Mouse, Hair, l(1)G0420, DmelCG12051, dmTAF[[II]]230, rat, protein polypeptide chains, act 88F, Cytoskeletons, Roles, Plastin 3 protein, Sn protein, Fascin, Concepts, Analysis, protein aggregate, present in fewer numbers in organism, Mass Spectrum Analysis, A, average, rsd, C, D, E, Fetal Hairs, cyt5C, YDR129C protein, plastin 1 (I isoform) protein, TFIID TAF250, Analyses, cel, M, vibrissa, hypoplasia, Swiss Mice, proteins, act88F, decreased, Fetal, act 42A, Role Concepts, Ac5C, CG4027, Act, s, Microfilaments, house mouse, DmelCG18290, act87E, I-PLASTIN protein, fascin-1, dTAF[[II]]230, l(1)G0330, Actin/BAP47, fascin protein, PLS1 protein, mouse, DFNB36, TAF200, act, Hairs, Normalcy, TAFII-250, TAF250/230, S pombe, Spectrum Analysis, act42A, Spectroscopy, HSN, ACTIN, TAFII250, Role Concept, fimbrin protein, AFFX-Dros-ACTIN_M_r_at, Mini-ICE, Role, actin, CG7478, Individual, CG5178, CG10067, Actin Microfilament, Act57A, Mus musculus, oligonucleotide random primer, whisker, Pls3 protein, ABP67 protein, DmelCG4027, Caspase-14 subunit p10, mice, l(1)G0117, Swiss Mouse, Spectrometry, Actin, Filaments, Caspase-14 subunit p19, CG17603, MICE, TAF[[II]], beta-actin/Bap47, Act57b, human, domesticus, l(1)G0486, act79B, retinal fascin, l(1)G0245, actin5C, Health, Drosophila, DMDA, l(1)G0009, Taf250, SR3-5, Mouse, singed protein, TAF230, act57b, Act5c, 3.4.22.-, d230, l(1)G0010, Act87e, fascin-2 protein, conformation, p55, ACT5C, CG12051, dTAFII250, mini-ICE, Spectrum Analyses, protein-containing complex, EfW1, anon-EST:CL2c12, Normalcies, TYPE, Stereovilli., act57B, DAGA4, CG30294, l(1)G0025, FSCN2 protein, act57A, Filament, Act88f, AI427122, reduced, polypeptide chain, dmTAF1, House, Taf230, subnumerary, actin-12, Mass, Mus musculus domesticus, tiny, MAM, plastin 3 (T isoform) protein, Mice, SCG3, je, RP30, Mass Spectroscopy, Lanugo, 143060_f_at, TAF250, Normalities, Taf200, act5C, dTAF[[II]]250, actin-bundling protein, Fetal Hair, DmelCG10067, Actin Microfilaments, cell, Swiss, coat/ hair, Pls1 protein, SNL, L-plastin, mus79B, SPBC1778.06c protein, Taf1p, Act57, decreased number, dTAF250, anon-EST:fe2D2, Act42a, fim1 protein, S cerevisiae, A930022G03, RANDOM, Actin Filaments, CG18290, Microfilament, fimbrin, quill, T10, liquid, T11, TAF, fascin homolog 2, Act-5C, relational structural quality, l(1)G0177, small, Ifm(3)7, fur, Individual Health, 42A, C630046B20Rik, TAF[[II]]250, SAC6 protein, act 79B, protein complex, I-plastin, l(3)84Ab, function, BG:DS00004.13, Stereovilli, BEST:RE38067, Cell, ahl8, LGMD2C, Concept, dTAF230, native protein, T-plastin, RFSN, natural protein, Mus, beta-actin, p230, utriculus, Protein, M32055, TAF[[II]]250/230, TFIID, Act42, Cytoskeleton, Mass Spectrum Analyses, protein p219, FAN1, Fan1, Random selection by shearing, T-fimbrin protein, Taf[[II]]250, l(1)G0079, Mass Spectrum, Stereovillus, Fscn2 protein, AI663989, TAF[[II]]230, fscn, DMDA1, Act88-F, underdeveloped, Normality, act 57A, retinal (Strongylocentrotus purpuratus) protein, hair, act 5C, House Mice, TAF[II]250, microchaeta, mus87E, plastin 1 (I-isoform) protein, Laboratory Mice, Actin Filament, DmelCG17603, Act(88F), SCARMD2, plastin 3 (T-isoform) protein, BAP47, coat hair, Bap47, Laboratory Mouse, Actin Cytoskeletons, Actin5C, TAF1Hair, mice., fur, setulae, whisker, Fetal Hairs, Fetal Hair, AI427122, quill, Fetal, Mus, coat/ hair, vibrissa, mouse, hair, Mouse, Hairs, house mouse, coat hair, Proteomes, microchaeta, Lanugo369trueComparison of hair bundle proteomes from WT and Pls1-knockout mouse utriclesWith their stereotyped dimensions and orderly arrangement, as well as their essential role in auditory and vestibular function, stereocilia of sensory hair cells provide a particularly prominent example of the importance of cellular actin protrusions. Uniformity of actin packing in stereocilia is mediated by actin crosslinkers of the plastin, fascin, and espin families. While mice lacking ESPN (espin) have no vestibular function, we found that mice that either lacked PLS1 (plastin 1) or had nonfunctional FSCN2 (fascin 2) had vestibular function that was modestly reduced, with double-mutant mice being more strongly affected. Despite its outsized importance in functional assays, targeted mass spectrometry indicated that ESPN only accounted for ~15% of the total crosslinkers in mouse utricle stereocilia. PLS1 was the most abundant of the crosslinkers, and the second-most-abundant protein overall in stereocilia. Stereocilia lacking PLS1 were shorter and thinner than wild-type stereocilia. Surprisingly, while wild-type stereocilia had random liquid packing of their actin filaments, stereocilia lacking PLS1 had orderly hexagonal packing. Stereocilia with normal PLS1 but that lacked functional FSCN2 had normal liquid packing and normal dimensions. All three crosslinkers are required for normal stereocilia structure and function, but PLS1 biases stereocilia towards liquid packing, which allows stereocilia to grow to greater 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