Characterisation of A2-24 glycan structure with sialic acid derivatisation. (a) Mass spectra of A2-24 species. The left panel shows the result of LC-ESI mass spectrometry (MS) analysis of the A2-24 fraction isolated by reversed-phase HPLC (see Fig. 4b). The product detected as a peak at m/z 1326.12 [M + 2 H]2+ was subjected to MS2 analysis and separated into several fragments as illustrated in the right panel. Constituents and possible linkage patterns for some products are also illustrated with symbols. NeuAc2Gal2GlcNAc3Man3FucGlcNAc2 (dN-BIBsF6 or dN-TRF6-1G) is annotated structure of strial glycan A2-24 (left panel). Note that the peak at m/z 884.63 in the left panel represents tri-protonated form [M + 3 H]3+ of the A2-24 glycan. (b) A full-scan positive ion mode MALDI-QIT-TOF mass spectrum (m/z > 2000) of the A2-24 fraction with sialic-acid-linkage–specific alkylamidation (SALSA). The strong signal at m/z 2755.11 [M + Na]+ represents the derivatised A2-24 species. The dotted line denotes the predicted position of the non-alkylamidated A2-24 glycan in sodiated form [M + Na]+ on the basis of its calculated m/z value (2672.97 marked by ‘Pre SALSA’). The difference in molecular mass between these two signals is shown (82.14 Da). (c) Assigned structure of the A2-24 strial glycan. Note that the linkage patterns among three N-acetylglucosamine residues in antenna structures and two mannose residues in the core structure have yet to be determined. PA: pyridylamine.