The workflow for characterisation of strial glycans. (a) An overview flowchart of the experiments conducted in this study. As the first step, glycans obtained from the stria vascularis were sequentially fractionated by three different HPLC types described in the orange box (upper left). The elution times recorded in reversed-phase and size fractionation HPLCs represent R and S values (Rstria and Sstria), respectively29,32 (blue box). In parallel, the original samples extracted via reversed-phase HPLC were analysed by positive ion mode LC-ESI-MS and MS2 (green box in the upper right); the spectra provided monosaccharide composition and linkage patterns. On the basis of these data, of the HPLC analyses of 194 standard glycans7,28, and of the empirical additivity rule7, we predicted set(s) of Rstd and Sstd values or those of Rcalc and Scalc values for each glycan (pink box). A comparison between this information and the values of Rstria and Sstria determined the structures of some glycan species (the deep-blue-filled ellipse). The other glycans whose structures were not completely determined by this procedure (the pale-blue-filled ellipse) were next subjected to SALSA and positive ion mode MALDI-MS (dark-purple box). This experiment revealed the sialyl linkage patterns of the majority of the analysed glycans (the pale-blue-filled ellipse). Nevertheless, characterisation of the sialyl linkage in A3-13 required a series of more complicated analyses consisting of SALSA and negative ion mode MALDI-MSn (pale-purple box). DEAE: diethylaminoethyl, HPLC: high performance liquid chromatography, LC-ESI: liquid chromatography with electrospray ionisation, MS: mass spectrometry, Rstd: R value of standard glycans, Sstd: S value of standard glycans, Rcalc: calculated R value, Scalc: calculated S value, SALSA: sialic-acid-linkage–specific alkylamidation, MALDI: matrix-assisted laser desorption/ionisation. (b) The numbers of strial glycans extracted or characterised by each series of the methods described in (a). The methods are shown on the left side. Of 107 glycans collected by reversed-phase HPLC, each of the 19 species marked by a single hash tag (#) was detected as a fraction whose peak area was less than 2% of that of the N-5 fraction in the subsequent HPLC chromatogram, and therefore each was excluded from further assays (see text). Glycans indicated by a double hash tag (##) were likely to be O-linked-type or non-specific moieties (see text). Groups highlighted in deep blue have glycans whose structures were determined perfectly, whereas groups coloured with pale blue consist of glycans whose partial linkages were assigned temporarily or those whose linkages were not completely clarified after the analyses. In summary, the structures of 79 strial N-glycans in total were profiled, as indicated by the black bar. Details of the experiments for A3-13 and A3-18 species are described in the main text. *Sialylated species that have glycosidic linkages inaccessible to the analyses using R and S values. **Species that were initially identified as a single sialylated glycan but later found to have an additional sialyl linkage isomer.