Analysis of A3-13 glycan structure with SALSA and multiple mass spectrometry modes. (a) A positive ion mode MALDI-QIT-TOF mass spectrum of A3-13 species. The sample analysed in this experiment was obtained by reversed-phase HPLC and derivatised with SALSA (see Supplementary Fig. S3). (b,c) Negative ion mode MALDI-QIT-TOF MS spectra of the reversed-phase HPLC fraction containing A3-13 species. The data on the samples without and with SALSA are displayed in panels b and c, respectively. In (b), the peaks at m/z 2316.8 [M-H]− and at m/z 2338.8 [M-H]− represent a deprotonated ion of the A3-13 strial glycan and its Na+-bound form, respectively, whereas the moiety (of the former) deficient in an acidic functional group is observed as a signal at m/z 2236.9 [M-H]−. Note that the deprotonated glycan (b) shifted by 41.1 Da with SALSA (c). (d) An MS2 spectrum of SALSA-derivatised A3-13 glycan isolated by the MS assay (a peak at m/z 2357.9 [M-H]− in (c). A signal at m/z 2025.7 [M-H]− should result from neutral loss of a single isopropylamine (iPA)-bound sialic acid in the parent ion. The lack of one and two HexNac residues likely produced two fractions at m/z 1822.6 [M-H]− and 1619.6 [M-H]−, respectively. (e) Data from the MS3 analysis of the product at m/z 1619.6 [M-H]− in MS2 mode. The signal at m/z 1174.3 [M-H]− stems from loss of the pyridylamine (PA)-bound GlcNAc and Fuc in the parent ion. The difference in m/z between this signal and other two peaks (m/z 971.2 [M-H]− and m/z 485.1 [M-H]−) can be explained by a loss of HexNAc or a loss of both HexNAc and three Hex residues, respectively, as shown in the panel. As described in the main text, the fraction at m/z 485.1 [M-H]− consists of GalNAcGlcNAc modified by a sulphate group. (f) The MS4 spectrum obtained from the parent ion detected as a signal at m/z 485.1 [M-H]− in MS3 mode. Difference in m/z value between the parent ion and the product at m/z 282.0 [M-H]− corresponds to the m/z value of a HexNAc residue. Composition of the moiety should be sulphated GalNAc or GlcNAc (see symbolic notations). (g) Possible structure of A3-13. This glycan has two GalNAc residues; however, which one is attached with a sialic acid or a sulphate group remains to be determined. (h) Mass spectra of A3-13 species (see also Supplementary Fig. S2). The left panel shows the result of LC-ESI MS analysis with the A3-13 fraction isolated through reversed-phase HPLC (see Fig. 4b). The fragment at m/z 1160.3 [M + 2 H]2+ was next subjected to a surgical MS2 assay, and the result is shown in right panel. Constituents and possible linkage patterns described above several peaks in both panels are based on the conclusion that A3-13 strial glycan is sulphated NeuAcGalNAc2GlcNAc2Man3FucGlcNAc2 [SO3−-6N-BI(dLdn1,2)F6] (see g). PA: pyridylamine.