Chapter 10: O-GalNAc Glycomics by LC–MS/MS

Mass spectrometry (MS) analysis of O-GalNAc glycans faces common glycomic challenges and presents unique considerations. Among the structural problems is the need to define the respective glycan chains and glycotopes on either the 6- or 3-arm of a branched core or those of an internal Gal attached to the reducing-end GalNAc. When considered together with the different positions of sialylation, fucosylation, and sulfation, the extent of isomeric variations can be too overwhelming to be resolved by any single analytical technique at the level of sensitivity and throughput demanded by current glycomics. Offline matrix-assisted laser desorption/ionization (MALDI)-based MS analysis is rather straightforward but ultimately limited by the quantitative identification of the isomeric constituents, particularly those of low abundance. Coupling to online liquid chromatography, either on a porous graphitized carbon column or on a reverse-phase C18 column for native and permethylated glycans, respectively, together with an MS²-product-dependent MS³ data acquisition workflow significantly increases the analytical depth. A good knowledge of the fragmentation pattern and diagnostic ions produced by different MS modes is indispensable to unambiguous structural assignments before any database-dependent, software-enabled data analysis can be routinely and reliably used. These technical aspects are highlighted and discussed at length here.