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

Abstract

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.

Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques

“…Additionally, glycosylated peptides often ionize poorly relative to non-glycosylated peptides [12]. In order to characterize a glycoproteome in detail, a MS workflow typically requires an efficient enrichment platform, a high-throughput MS analysis, and algorithms for targeted glycoproteomics [13,14]. To this end, a number of MS workflows have been established for confident discovery and identification of complex mixtures of glycopeptides [13–19].…”

Mass spectrometry for glycosylation analysis of biopharmaceuticals

“…More recently, IdeS and other enzymes for limited proteolysis generating larger peptides for a faster middle-up glycoprofiling were successfully applied for lot variability and biosimilar analysis of mAbs via LC-ESI-(Q)TOF-MS [44,50]. C 18 -RP materials commonly used for bottom-up proteomics may not retain certain glycopeptides due to increased analyte hydrophilicity compared to non-glycosylated peptides, requiring additional use of porous graphitize carbon (PGC) [41] or HILIC [51]. HILIC separation of glycopeptides is based on the properties of the peptide and the glycan moiety, and tolerates various substitutions, such as sulfation [52].…”

“…HILIC separation of glycopeptides is based on the properties of the peptide and the glycan moiety, and tolerates various substitutions, such as sulfation [52]. A recent approach demonstrated that on-line HILIC pre-enrichment of N-glycopeptides prior to C18-LC-QTOF-MS/MS resulted in efficient site-specific glycoprofiling [51]. The detection of both glycan and peptide fragmentation in the same MS/MS spectrum via elevated-energy CID allowed confirmation of both peptide and glycan sequence, and site assignment.…”

“…Though LC-ESI-IT-MS is widely used for glycopeptide analysis in biopharmaceuticals, LC-ESI-(Q)TOF-MS is gaining interest due to its higher resolution and mass accuracy [10,51,54,55].…”

Cancer serum biomarkers based on aberrant post-translational modifications of glycoproteins: Clinical value and discovery strategies

“…MS, combined with efficient separation techniques, has become a powerful tool for structural analysis of carbohydrates [59]. Some of its advantages include low sample consumption and high sensitivity.…”