N-glycan Extraction and Analysis Protocol

N-glycan extraction and purification of neutral and acidic N-glycans, and the mass spectrometric analysis was performed as earlier described^{18 (link),19 (link)}. Briefly, the glycans were enzymatically detached by N-glycosidase F (PNGase F) digestion from the deparaffinized tissue specimens (Glyko; ProZyme Inc., Hayward, CA). After the digestion, the N-glycans were purified by solid-phase extraction in 96-well format. The Bruker Ultraflex III TOF/TOF instrument (Bruker Daltonics Inc, Bremen, Germany) was used to perform matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry to the glycan samples. The mass spectrometry detected acidic N-glycans in negative ion reflector mode primarily as [M− H]¯ ions and neutral N-glycans in positive ion reflector mode primarily as [M+ Na]⁺ ions. Satomaa et al.^{20 (link)} and Saarinen et al.^{19 (link)} have previously described how the raw data was processed into presented glycan profiles. All the MS data are presented in Supplementary Tables S1 and S2.