Figure 2. Peptide MS-based identification of hair bundle-specific XIRP2 isoforms. A, Schematic depiction of the Xirp2 locus on chromosome 2 of the mouse genome. The two major XIRP2 isoforms are illustrated, with the exon usage highlighted by connecting lines to the genomic track: the canonical, long XIRP2 transcript is depicted above (lacks upstream exons −1 to −7, includes exon 7, and the frame of exon 8 encodes a stop codon, preventing translation of exon 9). The short Xirp2 transcript is shown below (including exons −7, −6 and −1, not present in long Xirp2; exons 1 and 7 are not present, but the frame of exon 8 permits translation through exon 9). B, Top left, Peptides from isolated utricular bundles that map to short XIRP2. Peptides that correspond to all exons predicted from transcript KM273012 (submitted to GenBank) were detected in the bundle data. Top right, Peptides isolated from utricular hair bundles that map to long XIRP2. Most peptides map to the N terminus of the long XIRP2 isoform; exon 7 has very few mapped peptides (arrow), suggesting that the long isoform is not present in the hair bundle. Bottom, Mouse peptides from GPMDB map to long XIRP2, including exon 7, and human peptides from GPMDB map to long XIRP2, including exon 7. C, Peptides detected in purified utricle bundles that span neighboring exons of short XIRP2. D, RT-PCR-based analysis of Xirp2 exon usage in heart and inner ear tissue: the transcript coding for the full-length short XIRP2 isoform was detected in inner ear, but absent in heart cDNA. The newly discovered 5′ exons (represented by a PCR fragment spanning exon −7 to exon 2) were nevertheless expressed in the heart, albeit at significantly lower levels compared with the ear. A PCR product indicative of the transition from exon 6 to exon 7 was detected in both ear and heart, but was stronger in the latter. A product indicative of the transition from exon 6 to 9 was detected in both ear and heart, but stronger in the former. E, Western blot analysis of hair bundles isolated from the mouse utricle (using the exon −6-specific antibody), detects a band with the mass of ∼105 kDa, consistent with the predicted mass of short XIRP2 (104.5 kDa).