LC-MS/MS-based structural glycoproteomics

Mass spectrometry (MS) is a fundamentally important tool for proteomics, providing protein sequences, quantification, as well as detailed information on post-translational modifications (PTMs). However, mass alone is not sufficient to solve the challenging problem of isomerism, the situation in which biologically distinct analytes have different structures but the same mass. An example of this can be found in leucine and isoleucine, which share a mass but are distinct amino acids.

Isomerism is a particular issue in the field of glycosylation analysis. Glycans ubiquitously cover our cells and proteins, often being the first barrier of inter-cellular communication and pathogen-host interactions, as well as being the most-accessible biomarker reservoir in a spatial sense. Glycosylation has long been studied by mass spectrometry, e.g., the glycans themselves after enzymatic release (glycomics), or the glycopeptides that result from proteolytic digestion (glycoproteomics). However, while glycans have a multitude of isomeric properties, including fucose position, sialic acid linkage and antennary branching structures, these are not readily determinable by MS. For released glycans, this can be partially overcome by orthogonal means, e.g., ion mobility, liquid chromatography (LC) using porous graphitic carbon, or chemical derivatization, but for glycopeptide analysis these techniques are not directly compatible. At the same time, only at the glycopeptide level can glycans reliably be positioned at given glycoproteins and glycosylation sites.

As such, to enable structural glycoproteomics, we have developed a combination of MS/MS-based structural glycopeptide characterization and high-resolution nano-HILIC-LC (Figure 1). As we demonstrate on a growing number structurally-defined glycopeptide standards, oxonium ion ratios follow a distinct path through collision-energy-space depending on the structural characteristics (Maliepaard, et al., 2023, Anal. Chem.). While this itself can already be used to quantify isomer ratios from a mixture, it also allows the structural characterization of glycopeptide isomers that show chromatographic separation, for which we have optimized HILIC-LC. By applying the resulting LC-MS/MS methodology on complex samples such as human plasma and recombinant IgGs, we reveal, for instance, a distinct branch asymmetry present amongst the IgG subclasses: galactosylation of IgG1 dominantly occurs at the 6-branch, opposite to IgG2 for which it is the 3-branch instead. Structural glycoproteomics is expected to yield similarly remarkable observations for other proteins and their interactions, to lead to improved drug design, as well as to significantly deepen the pool of glycosylation-based biomarkers.