Flexcontrol 3.0 software package

Samples for MALDI-TOF-MS were prepared from 10 μg of purified IgGs from CIA-induced ST6Gal1^f/f AID-Cre, ST6Gal1^f/f, AID-Cre and C57BL/6j mice and total serum IgG from ST6Gal1 KO mice by BlotGlyco (Sumitomo Bakelite Co., Tokyo, Japan) according to the manufacturer's protocol55 (link). Samples were analysed by MALDI-TOF system by using an Autoflex III TOF/TOF mass spectrometer equipped with a reflector and controlled by the FlexControl 3.0 software package (Bruker Daltonics GmbH, Bremen, Germany). Details of the method used for MALDI-TOF-MS analysis are described in Supplementary Methods.

The harvested culture cells and the EVs were suspended in acetate buffer (50 mM, pH 5.5) containing 0.2% Triton X-100 and sonicated with an Ultrasonic Homogenizer (TAITEC, Saitama, Japan). To release GSL-glycans, 5 μL of endoglycoceramidase I (EGCase I) from Rhodococcus equi was added to their lysates of cultured cells and their EVs corresponding to 40 μg protein^{47 (link)}. EGCase digestion was performed at 37 °C for 16 h. After deglycosylation, ethanol was added to the reaction mixture and incubated at −30 °C for 4 h. For recovery of GSL-glycans, the supernatant fractions were separated by centrifugation and dried with a centrifugal evaporator. The concentrated supernatant containing GSL-glycans were resuspended in 50 μL of H₂O and subjected to a glycoblotting procedure as previously described^{50 (link)}. The analysis of GSL-glycans was performed by MALDI-TOF MS using an Ultraflex II TOF/TOF mass spectrometer, which was controlled by the FlexControl 3.0 software package (Bruker Daltonics, Bremen, Germany). All spectra were obtained as positive ions and masses were annotated using the FlexAnalysis 3.0 software package (Bruker Daltonics). The SphinGOMAP (http://www.sphingomap.org/) online databases were used for structural identification of GSL glycans^{51 (link)}.