Fig. 5: Chromatograms and structural assignments of Hex₈GlcNAc₂ N-glycan from soybean and Man₄GlcNAc₂ N-glycans from hen egg ovalbumin.

a Chromatograms (total intensity of fragments from ion m/z 1743) of Hex₈GlcNAc₂ N-glycan extracted from soybean proteins. This chromatogram shows the last separation in two-dimensional high-performance liquid chromatography (HPLC). b Chromatograms of the eluents collected from the chromatogram in (a). The eluents corresponding to the chromatogram in (a) were collected every 30 s. After repeating the collection of ten times, the eluents were stored in collecting tubes at room temperature for 6 h. Then the eluents were concentrated and reinjected into the same HPLC separately. The same retention time and relative intensities of the two peaks in chromatogram (b) are the same as that in chromatogram (a), representing they represent the α and β anomeric configurations of the sugar at the reducing end of the same isomer. c Chromatogram (total intensity of fragments from ion m/z 1097) of Man₄GlcNAc₂ N-glycans extracted from hen egg ovalbumin. This chromatogram shows the last separation in four-dimensional HPLC. The reduction of Man₄-N-glycans was performed to demonstrate that LODES/MSⁿ can be applied to N-glycans after reduction. Two peaks in the chromatogram represent two isomers. The details of HPLC conditions are described in Methods and the mass spectra used in LODES/MSⁿ for structural determination are presented in Supplementary Information Figs. S4 and S5.