Glycan Profiling Using glyXboxCE

To evaluate the performance of non-enzymatic approaches for OSI depletion and the progress of enzyme reactions, glyXboxCE™ (glyXera, Magdeburg, Germany) was used, including the sample preparation kit glyXprepCE™. Briefly, bovine ribonuclease B, fetuin, and IgG as standard glycoproteins and stem cell lysates were dissolved in PBS and de-N-glycosylated by peptide-N-glycosidase F according to the kit instructions. Maltodextrins and dextrans were dissolved in ultrapure water. Maltodextrins, dextrans, and released N-glycans were labeled with APTS and purified by hydrophilic interaction chromatography (HILIC) in solid phase extraction (SPE) mode as described in the kit instructions. APTS-labeled analytes were analyzed with xCGE-LIF using glyXboxCE™. Obtained data was processed using glyXtoolCE™ (glyXera GmbH, Magdeburg, Germany) [73 ]. The software performed a migration time alignment of electropherograms to an internal standard, resulting in aligned N-glycan fingerprints. Peaks with a signal-to-noise ratio >10 were picked, and relative peak intensities were calculated based on peak height normalization. N-glycan structures were determined with the glyXtoolCE™ integrated N-glycan database and confirmed with exoglycosidase digests (see Supplementary Methods).