In this chapter, we will present two methods for comprehensive glycoprotein characterization that are particularly but not exclusively useful for Pichia pastoris glycoproteins. One approach is intact protein mass measurement, where deglycosylation may be used to determine the mass of the unmodified protein. The other method is the classical bottom-up approach, where peptides and glycopeptides are analyzed by reversed-phase chromatography and detected by electrospray ionization mass spectrometry. The choice of chromatography solvents with a high ionic strength simplifies the identification of peaks of a particular peptide's glycopattern as it leads to co-elution of neutral and charged, i.e., phosphorylated, glycoforms.
Keywords: Ammonium formate buffer; Electrospray ionization (ESI); Mass spectrometry; Monolithic protein column; Phosphorylation; Reversed-phase chromatography.