APPLICATIONS OF ELECTROSPRAY MASS-SPECTROMETRY TO ERYTHROPOIETIN N-LINKED AND O-LINKED GLYCANS

The improved sensitivity and soft ionization characteristics of electr ospray (ES) mass spectrometry (MS) has been applied to the glycan stru ctures 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 der ivatives. The O-linked glycopeptide was also analyzed directly. In thi s fraction seven glycans were identified (two previously unreported) i n addition to the unglycosylated peptide. The three N-linked fractions were divided with one fraction preceded by periodate oxidation and re duction to resolve isomeric structures, linkage, and branching pattern s, and confirm the overall structural motif. All were methylated and p rofiled. Two biantennary, 8 triantennary, and 10 tetraantennary struct ures were identified with approximately 13% of each N-linked glycan po ssessing a single N-glycolylneuraminyl analog. The minimal energies of ionization, the absence of a matrix background, and enhanced sensitiv ity brings an improved technology for studying carbohydrate structural detail. (C) 1994 Academic Press, Inc.