Reproducible and sensitive detection of simple and complex glycan isomers using a new porous graphitized carbon phase packed in micro-bore geometries

Porous graphitized carbon (PGC) LC-MS/MS-based detection of glycans ranging from approximately 3-15 saccharide residues is a gold standard in glycomics; however, robust glycoprofiling workflows that also cover mono- and disaccharide glycans (e.g. Tn, T, sialyl Tn O-glycans) are less mature. Given the growing appreciation that short glycans are biologically and clinically important, we here address this analytical shortcoming by testing the glycoprofiling performance of a new Supel Carbon PGC phase. Employing a shallow gradient of organic solvent, we systematically explored the separation potential of two micro-bore column geometries (15 cm x 0.3 mm/1 mm) using mixtures of isobaric monosaccharides (GlcNAc, GalNAc), disaccharides (lactose, maltose) and complex N- and O-glycans from pig gastric mucin, bovine submaxillary mucin, THP-1 cells, bovine fetuin and recombinant human IgM. Matching an existing PGC phase (HyperCarb), the Supel Carbon columns consistently retained isobaric mono- and disaccharides, but afforded improved baseline separation enabling confident saccharide quantitation. The Supel Carbon PGC columns also efficiently separated short O- and N-glycans released from biological sources including disease-relevant Tn, T and sialyl Tn while maintaining effective separation of elongated and branched glycans including near-identical structural isomers. Reproducible glycan detections were achieved within and across column manufacturing lots and low femtomolar detection limits were observed for the micro-bore columns. Finally, the impacts of alternative organic solvents (methanol, ethanol) and flow rates on column performance were also established. Our findings position the Supel Carbon PGC phase as a valuable tool in the glycomics toolbox enabling reproducible profiling of truncated and elongated glycan isomers.