Applications of Electrospray Mass Spectrometry to Erythropoietin N- and O-Linked Glycans

The improved sensitivity and soft ionization characteristics of electrospray (ES) mass spectrometry (MS) has been applied to the glycan structures of recombinant erythropoietin (rEPO). The four glycopeptides (O-126, N-24, N-38, N-83) were mapped, isolated, deglycosylated, and the glycans profiled (without desialylation) by ES-MS as their methyl derivatives. The O-linked glycopeptide was also analyzed directly. In this fraction seven glycans were identified (two previously unreported) in addition to the unglycosylated peptide. The three N-linked fractions were divided with one fraction preceded by periodate oxidation and reduction to resolve isomeric structures, linkage, and branching patterns, and confirm the overall structural motif. All were methylated and profiled. Two biantennary, 8 triantennary, and 10 tetraantennary structures were identified with approximately 13% of each N-linked glycan possessing a single N-glycolylneuraminyl analog. The minimal energies of ionization, the absence of a matrix background, and enhanced sensitivity brings an improved technology for studying carbohydrate structural detail.