O- and N-glycosylation analysis of cell lines by ultrahigh resolution MALDI-FTICR-MS

Glycosylation analysis from biological samples is often challenging due to the high complexity of the glycan structures found in these samples. In the present study N- and O- glycans from human colorectal cancer cell lines and human plasma were analyzed using ultrahigh resolution MALDI-FTICR-MS. N-glycans were enzymatically released from cell lines and plasma proteins, whereas beta-elimination was used for the release of O-glycans from the cells. The purified samples were mass analyzed using a 15T MALDI-FTICR-MS system, with additional MS/MS (collision-induced dissociation) experiments for O-glycan identifications. A total of 104 O-glycan and 62 N-glycan compositions were observed in the spectra obtained from colorectal cancer cell line samples. In the cell line N-glycan spectra, the highest intensity signals originated from high-mannose glycans, next to the presence of various complex type glycans. Notably, in the O-glycan spectra mono- and disaccharide signals were observed, which are difficult to detect using alternative glycomic platforms such as porous graphitized carbon LC-MS. In the N-glycan spectra from plasma, isobaric species were resolved in MALDI-FTICR-MS spectra using absorption mode whereas these overlapped in magnitude mode. The use of ultrahigh resolution MALDI-FTICR-MS for the analysis of glycans in complex mixtures enables us to confidently analyze glycans in the matrix region of the spectrum and to differentiate isobaric glycan species. •MALDI-FTICR-MS allows profiling of released N-glycans and permethylated O-glycans from cell lines.•Ultrahigh resolution MS results in high confidence identification of glycans with compositional assignment.•Absorption mode analysis of MALDI-FTICR data results in ultrahigh resolution glycan signals preventing many interferences.•Permethylated O-glycan alditols ranging from monosaccharide to large oligosaccharides are covered in a single MS spectrum.