Select series cyclic ion mobility

The HPLC–MS measurements were performed on a Waters Acquity I-class UPLC instrument (Milford, MA, USA) coupled to a Waters Select Series Cyclic Ion Mobility (Milford, MA, USA) mass spectrometer. For the chromatographic separation of CS and HS disaccharides, a self-packed GlycanPac AXH-1 capillary column (250 µm i.d.) was used with the ammonium formate salt gradient methods published before [17 (link),18 (link)]. In the low-flow ESI ion source, the capillary voltage was set to 1.9 kV, while the cone voltage was 20 eV, and the temperature was 120 °C. The HS disaccharides were measured in MS1 mode, with the trap collision energy being 6 eV, and the transfer being 3eV. The CS was measured in MS1 and MS/MS modes, where the monosulfated isomer pairs were fragmented with 32 eV in the transfer to determine sulfation positions. Finally, the extracted ion chromatograms were integrated with the TargetLynx add-in of MassLynx software v4.2, Waters Corporation (Milford, MA, USA). The detailed integration method parameters are summarized in Table S2. Chromatogram examples are shown in Appendix A: Figure A1 (representative extracted ion chromatograms of CS disaccharides) and Figure A2 (representative extracted ion chromatograms of HS disaccharides).

The HPLC–MS measurements were performed on a Waters Acquity I-class UPLC (Milford, MA) coupled to a Waters Select Series Cyclic Ion Mobility (Milford, MA) mass spectrometer. For the chromatographic separation of CS/DS and HS disaccharides, a self-packed GlycanPac AXH-1 capillary column (250 μm i.d.) was used with the ammonium formate salt gradient methods published before [23 (link), 24 (link)]. In the low-flow ESI ion source, the capillary voltage was set to 1.9 kV, while the cone voltage was 20 eV and temperature was 120 °C. The HS disaccharides were measured in MS1 mode, the trap collision energy being 6 eV and the transfer being 3 eV. The CS/DS was measured in MS1 and MS/MS mode, where the monosulfated isomer pairs were fragmented with 32 eV in the transfer to determine stereochemistry.