(A) Shotgun mass-spectrometry quantitation of 3313 proteins detected in at least three of five purification fractions where enrichment of stereocilia membranes was expected. The slope of the purification fraction (assuming an interval of 1) vs. log riBAQ was calculated for each protein, and the top 25 proteins with the steepest positive slopes ('enrichment slope') were highlighted. Color indicates the slope steepness. For this analysis, ATP2B2 was split out of the group also containing ATP2B1 and ATP2B4 group; data corresponding both ATP2B2 and PTPRQ were emphasized to illustrate the effectiveness of the stereocilia-membrane enrichment. The number of proteins detected in each fraction was also indicated. (B) Gene ontology analysis with DAVID of the S7 (RIPA-soluble) fraction using S1 (PNS) as background. The top ten cellular component terms enriched are indicated; the log of the P-value (adjusted for false-discovery rate, FDR) is plotted. The inset shows a magnification of the 'plasma membrane' term, which had a substantial increase in significance in the S7 fraction. (C) Relationship between enrichment slope and frequency of Ensembl-annotated transmembrane helicies. Each protein identified was assigned 1 if they were annotated as having a transmembrane helix, and 0 if not. All proteins were sorted according to enrichment slope, and an average was calculated every 50 proteins. (D) Stereocilia membrane protein enrichment. Displayed are bundle-enriched (>three-fold) proteins from Wilmarth et al. (2014) that were annotated as containing transmembrane domains, plus SLC34A2, SLC9A9, and SLC9A6. ND, not detected. (E) Clustering analysis of shotgun mass-spectrometry data using mclust. After data were log-transformed, they were normalized within a row, subtracting the row mean and dividing by the row standard deviation (thus the resulting mean was 0 and the standard deviation was 1). The best solution returned 25 clusters, which were ordered here by the enrichment slope (only the five fractions used in A were used to calculate the slope). PTPRQ, ATP2B2, and XIRP2 were all in the cluster with the steepest enrichment slope. (F–I) Targeted proteomics analysis of enrichment fractions. (F) Chromatograms of SAPLHILTDEDAPSSPPESLSVK peptide from PTPRQ detected in S1 (dark gray) and S7 (orange) fractions; inset shows magnified S1 chromatogram. Sum of 12 daughter ions. Arrows indicate where multiple MS2 spectra matched to PTPRQ were acquired. (G) Example MS2 spectrum from S7 sample; multiple peaks matched to predicted PTPRQ peptide fragment ions. (H) Targeted proteomics analysis of enrichment fractions. The mean of the total intensity area of 4–8 peptides per protein is indicated. (I) Same data as G, except area relative to S1 is indicated.