Comprehensive Glycan Mapping of Intact Human Erythropoietin by Reversed-Phase Liquid Chromatography-Mass Spectrometry

We report a method for the nearly complete characterization of glycoforms expressed in human erythropoietin (EPO_HUMAN) using mass spectrometry. The method involves reversed-phase LC-MS analysis of intact glycoproteins, top-down sequencing of the protein backbone, and bottom-up LC-MS/MS of its digests using electron capture dissociation (ECD) and collision-induced dissociation (CID). In the top-down sequencing by ECD, the loss of the C-terminal arginine residue was confirmed. Using the protease Glu-C, the bottom-up approach yielded site-specific glycan structures, including sialic acid linkages. We also applied ECD to di-N-glycosylated peptides that appeared in the tryptic digest to resolve ambiguities in the assignments of glycans between two neighboring N-glycosylation sites. We reconstructed nearly 100 intact glycoforms by assembling the obtained information. Interestingly, the N-glycosylation site N₅₁ predominantly exhibited a wide variety of glycan structures, including nonglycosylated, GalGlcNAc-type antennary, and GalNAcGlcNAc-type antennary fucosylated N-glycans, as well as phosphorylated high-mannose N-glycans that were not fucosylated.