The phospholipid- and Ca2+-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear's sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, shotgun and targeted mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5-/- mice. Annexins have been proposed to mediate Ca2+-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, Anxa5-/- mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle's key function, mechanotransduction. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance.

For targeted MS/MS, we carried out in-solution tryptic digests of hair-bundle samples using the enhanced filter-aided sample preparation (eFASP) method (Erde et al, J Proteome Res 2014). Proteins were digested with 200 ng sequencing-grade modified trypsin (Promega) in the filter unit; a total volume of 100 µl digestion buffer was used and the reaction was carried out at 37°C for 12-16 hours. After isolating peptides by centrifugation, we extracted them with ethyl acetate to remove remaining deoxycholic acid. Peptide samples were analyzed with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) coupled to a Thermo/Dionex Ultimate 3000 Rapid Separation UPLC system and EasySpray nanosource. Samples were loaded onto an Acclaim PepMap C18, 5 μm particle, 100 μm x 2 cm trap using a 5 μl/min flow rate; peptides were separated on a EasySpray PepMap RSLC, C18, 2 μm particle, 75 μm x 25 cm column at a 300 nl/min flow rate. Solvent A was water and solvent B was acetonitrile, each containing 0.1% (v/v) formic acid. After loading at 2% B for 5 min, peptides were separated using a 55-min gradient from 7.5-30% B, 10-min gradient from 30-90% B, 6-min at 90% B, followed by a 19 min re-equilibration at 2% B. Peptides were analyzed using the targeted MS2 mode of the Xcalibur software in which the doubly or triply charged precursor ion corresponding to each peptide was isolated in the quadrupole, fragmented by HCD, and full m/z 350-1600 scans of fragment ions at 30,000 resolution collected in the Orbitrap. Targeted MS2 parameters included an isolation width of 2 m/z for each precursor of interest, collision energy of 30%, AGC target of 5 x 104, maximum ion injection time of 100 ms, spray voltage of 2400 V, and ion transfer temperature of 275°C. No more than 75 precursors were targeted in each run and no scheduling was used. Two to five unique peptides for each protein of interest were chosen for isolation based on previous data-dependent discovery data or from online peptide databases (www.peptideatlas.org, www.thegpm.org). We used the software package Skyline (http://proteome.gs.washington.edu/software/ skyline/) to generate precursor isolation lists for all peptides of interest and export them into the Orbitrap control software. Skyline was used to analyze targeted MS/MS data. Chromatographic and spectral data from RAW files were loaded into Skyline and manually analyzed to identify fragment ion peaks corresponding to each peptide. RAW files were also processed using Proteome Discoverer (Thermo Scientific) software in order to match MS/MS spectra to an Ensembl spectral database using Sequest HT. Fragment ion peaks for each peptide were chosen according to the following criteria: 1) three or more co-eluting fragment ions contributed to the peak signal, 2) two or more data points were collected across the peak, and 3) one or more spectrum within the peak were matched to correct peptide sequence within the spectral database. If spectra within a specific sample were not identified then a) the retention time of the chosen peak must be within 2 minutes of the retention time of an identified peak for that peptide from another sample and b) the type of daughter ions contributing to the peak must match the identified peptide peak from another sample. If no peak matching these criteria was found in a particular sample the peak area was counted as zero. Chromatographic peak areas from all detected fragment ions for each peptide were integrated and summed to give a final peptide peak area. The peptide peak areas for each protein of interest were averaged for each sample, then averaged for each protein of interest across the biological replicates. The Student's t-test was used to determine statistical significance between conditions (wild-type vs. Anxa5-/- mice, or Ca2+-containing vs. no added Ca2+ buffer solutions).

Hair bundles were isolated using the twist-off method, as adapted for mouse utricle (Krey et al, Sci Data 2015). The utricles were adhered to 35 mm plastic dishes (untreated EASY GRIP Falcon Petri dishes; Becton Dickinson) in Leibovitz's L-15 Medium without phenol red (21083-027; Thermo Life Technologies). This medium contains 1.26 mM CaCl2. After removing otolithic membranes with an eyelash, a plastic washer was placed around the utricles; 4.5% low-melting point agarose in L-15 at 42°C was added. After the agarose was set at 4°C for 5-10-20 min, the utricles were removed from the agarose, leaving bundles in the agarose. To clear away obvious cellular debris, a tungsten needle was used to cut away blocks of agarose; bundles from a single utricle were removed in <0.5 µl agarose plug. Isolated bundles in agarose were frozen at -80°C, and were pooled later for mass spectrometry analysis. For experiments isolating bundles under low Ca2+ conditions, HBSS (14025-076; Life Technologies; 1.26 mM CaCl2 ) and HBSS without calcium and magnesium (SH30588.02; HyClone) solutions was were substituted for L-15. For the WTvsKO dataset, each sample consisted of hair bundles isolated from 10 utricles from either wildtype or Anxa5 -/- mice. Three biological replicates for each genotype were run. For the Ca2+vsNoCa2+ dataset, each sample consisted of hair bundles isolated from 5 utricles dissected from wildtype mice in either uM or mM levels of calcium.