Confident Assignment of Site-Specific Glycosylation in Complex Glycoproteins in a Single Step

A glycoprotein may contain several
sites of glycosylation, each
of which is heterogeneous. As a consequence of glycoform diversity
and signal suppression from nonglycosylated peptides that ionize more
efficiently, typical reversed-phase LC–MS and bottom–up
proteomics database searching workflows do not perform well for identification
of site-specific glycosylation for complex glycoproteins. We present
an LC–MS system for enrichment, separation, and analysis of
glycopeptides from complex glycoproteins (>4 N-glycosylation
sequons) in a single step. This system uses an online HILIC enrichment
trap prior to reversed-phase C18-MS analysis. We demonstrated the
effectiveness of the system using a set of glycoproteins including
human transferrin (2 sequons), human alpha-1-acid glycoprotein (5
sequons), and influenza A virus hemagglutinin (9 sequons). The online
enrichment renders glycopeptides the most abundant ions detected,
thereby facilitating the generation of high-quality data-dependent
tandem mass spectra. The tandem mass spectra exhibited product ions
from both glycan and peptide backbone dissociation for a majority
of the glycopeptides tested using collisionally activated dissociation
that served to confidently assign site-specific glycosylation. We
demonstrated the value of our system to define site-specific glycosylation
using a hemagglutinin containing 9 N-glycosylation
sequons from a single HILIC-C18-MS acquisition.