Glycoform characterization of intact erythropoietin by capillary electrophoresis-electrospray-time of flight-mass spectrometry

Glycosylated proteins often show a large variation in their glycosylation pattern, complicating their structural characterization. In this paper, we present a method for the accurate mass determination of intact isomeric glycoproteins based on capillary electrophoresis-electrospray-time of flight-mass spectrometry. Human recombinant erythropoietin has been chosen as a showcase. The approach enables the on-line removal of nonglycosylated proteins, salts, and neutral and negatively charged species. More important, different glycosylation forms are separated both on the base of differences in the number of negatively charged sialic acid residues and the size of the glycans. Thus, 44 glycoforms and in total about 135 isoforms of recombinant human erythropoietin, taking also acetylation into account, could be distinguished for the reference material from the European Pharmacopeia. Distinct glycosylation differences for samples from different suppliers are clearly observed. Based on the accurate mass an overall composition of each single isoform is proposed, perfectly in agreement with data on glycan and glycopeptide analysis. This method is an ideal complement to the established techniques for glycopeptide and glycan analysis, not differentiating branching or linkage isoforms, but leading to an overall composition of the glycoprotein. The presented strategy is expected to improve significantly the ability to characterize and quantify isomeric glycoforms for a large variety of glycoproteins.