Deep structural analysis and quantitation of O-linked glycans on cell membrane reveal high abundances and distinct glycomic profiles associated with cell type and stages of differentiation.

Proteins on cell membrane are modified by N- and O-glycans. N-Glycans have been extensively characterized using advanced separation and mass spectrometry techniques. However, O-glycans remain a challenge due to the lack of universal enzyme to release them and the large background abundances of N-glycans. Here, we report a method for in-depth structural analysis and quantitation of O-glycans derived from human cell membrane. O-Glycans were chemically released from isolated cell membrane glycoproteins following N-glycan and lipid/glycolipid removal by PNGase F digestion and Folch extraction, respectively. Released O-glycans were purified by an optimized protocol to eliminate interference from small molecules and degraded proteins. Cell surface O-glycans were then analyzed using a nanoLC-chip-QTOF mass spectrometer with a porous graphitized carbon (PGC) column while the N-glycans and glycolipids isolated from the same cell membrane fractions were analyzed in parallel using previously reported methods. The monosaccharide compositions and linkages of the detected O-glycans were identified by exoglycosidase digestion facilitated with tandem MS. Using this method, we identified 44 cell membrane O-glycan isomers with tandem MS, and among them we unambiguously characterized 25 O-glycan structures with exoglycosidase digestion to create a library with their complete structures, accurate masses and retention times. In this process, we identified and characterized unexpected mannose oligomers that are α(1-2/3) linked. This library enabled the identification and quantification of unique cell surface O-glycans from different cell lines and the study of specific O-glycan changes during cell differentiation.