Extended Data Fig. 5: Analysis of background ions detected in full MS indicates that the mucin glycan pools do not contain detectable peptide contaminants.

O-glycan pools released (Methods) from mucin glycoproteins (MUC5B is shown as an example) were permethylated and analyzed by NSI-MS following porous graphitized carbon (PGC) clean-up. a) Full MS profile of a mixture of permethylated MUC5B O-glycans. Magnified view of lower mass range (m/z = 200-430) is highlighted in subsequent panels. b) Ions detected in range m/z = 200-430 following injection of vehicle (pure methanol) to demonstrate solvent background. c) Ions detected in range m/z = 200-430 following injection of permethylated MUC5B O-glycans (magnification of spectrum in panel a). Note largest peaks in this mass range are less than 5% of the signal intensity of the largest peaks in panel a. d) Ions detected in range m/z = 200-430 following injection of permethylated N-linked glycans released from a tryptic digest of HEK cell glycoproteins by PNGaseF and subsequent Sep-Pak C18 clean-up, which is unlikely to contain any peptide contamination. The low-abundance contaminant peaks detected in the HEK N-glycan preparation are at m/z values equivalent to those detected in the mucin O-glycan preparation and are, therefore, not derived from contaminant peptides or glycopeptides produced by hydrazinolysis. Y-axis is shown as absolute intensity.