Hydrophilic Interaction Chromatography Coupled to Ultraviolet Photodissociation Affords Identification, Localization, and Relative Quantitation of Glycans on Intact Glycoproteins

Protein glycosylation is implicated in a wide array of
diseases,
yet glycoprotein analysis remains elusive owing to the extreme heterogeneity
of glycans, including microheterogeneity of some of the glycosites
(amino acid residues). Various mass spectrometry (MS) strategies have
proven tremendously successful for localizing and identifying glycans,
typically utilizing a bottom-up workflow in which glycoproteins are
digested to create glycopeptides to facilitate analysis. An emerging
alternative is top-down MS that aims to characterize intact glycoproteins
to allow precise identification and localization of glycans. The most
comprehensive characterization of intact glycoproteins requires integration
of a suitable separation method and high performance tandem mass spectrometry
to provide both protein sequence information and glycosite localization.
Here, we couple ultraviolet photodissociation and hydrophilic interaction
chromatography with high resolution mass spectrometry to advance the
characterization of intact glycoproteins ranging from 15 to 34 kDa,
offering site localization of glycans, providing sequence coverages
up to 93%, and affording relative quantitation of individual glycoforms.