Hydrophilic interaction liquid chromatography-mass spectrometry for the characterization of glycoproteins at the glycan, peptide, subunit, and intact level

Abstract This chapter presents a methodical overview of recent research literature dealing with the characterization of glycoproteins by hydrophilic interaction chromatography separations coupled to mass spectrometry (HILIC-MS). This includes the analysis of intact glycosylated proteins as well as released glycans, glycopeptides, and protein subunits obtained after enzymatic digestion. The chapter provides basic background information on HILIC and glycoproteins, and the molecular levels at which glycosylation can be characterized. Next, essential HILIC parameters such as (the choice of) stationary phase type, mobile phase conditions, injection volume, and column temperature are reviewed. Herein focus is on the effect that these parameters have on both HILIC separation and MS detection. This is discussed in the context of the respective levels (glycan/peptide/subunit/intact) of glycoprotein analysis and current trends are highlighted. In the final part of this chapter, these developments are put in perspective by treating several applications of HILIC-MS for glycoprotein analysis in more detail, particularly in biopharmaceutical and clinical fields.