General procedures for glycan release

Detailed sample preparation procedures for glycomics analysis by mass spectrometry have been described previously⁶³. In brief, frozen human lung tissue was homogenized and sonicated in ice-cold ultra-pure water. Glycolipids were extracted by the addition of methanol and chloroform whereas glycoprotein precipitants were collected by centrifugation. Glycans were released from GSLs via a recombinant endoglycoceramidase II (rEGCase II; Takara Bio USA Inc.) and reduced with sodium borohydride. Glycopeptides were reduced and carboxymethylated with dithiothreitol and iodoacetamide, followed by overnight trypsin digestion. N-linked glycans were released from glycopeptides by PNGase F and O-linked glycans by reductive elimination. All glycans were permethylated, chloroform extracted and purified by C18 Sep-Pak pre-packed columns (WAT054945, Waters Corp.) prior to mass spectrometric analysis.