Targeted glycomics

The complexity of glycosylation severely limits the usefulness of untargeted approaches for quantitative glycomics and glycoproteomics. In addition, glycoproteomics suffers from the fact that peptides generally ionize better than glycopeptides in MALDI- and ESI-MS. Therefore, we have a number of protocols to purify a single protein or a subset of proteins from complex samples by affinity purification.

Immunoglobulins and Fc vs. Fab

We frequently assess immunoglobulin glycosylation in serum/plasma and other complex samples. High throughput affinity purification protocols are available to isolate immunoglobulin G (IgG) and immunoglobulin A (IgA).1,2 A well-controlled tryptic digestion step is at the core of a robust protocol. Subclass specific Fc-glycosylation profiles for IgG are obtained in a robust manner by tight control of the analytical process, for example the tryptic cleavage step.3 Alternatively, Fc- and Fab-glycosylation of IgG can be measured in parallel using a combination of IdeS digestion and glycan release.4 IgG glycosylation is measured either by LC–MS or MALDI-MS. For IgA, differential profiles for the two conserved N-glycosylation sites and hinge region O-glycosylation are obtained by MALDI-FTICR-MS.2

N-glycomic analysis of IgG Fab glycosylation and Fc glycosylation. This workflow has been applied to study IgG glycosylation changes with pregnancy4 and arthritis (manuscript submitted)

Depletion and abundant serum glycoproteins

Human plasma is a complex matrix containing hundreds of glycoproteins being potential biomarkers for diseases, for example congenital disorders of glycosylation. As data obtained from glycoproteomic studies of human plasma is often dominated by high abundant proteins and information about low abundant proteins remains challenging due to the wide range of protein concentrations.5

Sample complexity is reduced and sensitivity towards less abundant proteins is enhanced, by depleting the fourteen most abundant plasma proteins. An affinity purification protocol using camelid antibody domains immobilized on agarose beads has been developed to this end. This enables sensitive LC-MS or CE-MS analysis and future automation of the protocol is envisioned.6 Samples can be successfully depleted by our method and more low-abundant proteins can be observed in the depleted fraction than in raw plasma. LC-MS/MS measurements identify 5 times more peptides compared to non-depleted plasma. Further performance improvements should ultimately enable N-glycosylation profiling of low abundant plasma proteins.

  1. Fokkink, W. J. et al. IgG Fc N-glycosylation in Guillain-Barre syndrome treated with immunoglobulins. J. Proteome Res. 13, 1722-1730 (2014).
  2. Bondt, A. et al. Longitudinal monitoring of immunoglobulin A glycosylation during pregnancy by simultaneous MALDI-FTICR-MS analysis of N- and O-glycopeptides. Sci. Rep. 6, 27955 (2016).
  3. Falck, D. et al. Glycoforms of Immunoglobulin G Based Biopharmaceuticals Are Differentially Cleaved by Trypsin Due to the Glycoform Influence on Higher-Order Structure. J. Proteome Res. 14, 4019-4028 (2015).
  4. Bondt, A. et al. Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes. Mol. Cell. Proteomics 13, 3029-3039 (2014).
  5. Clerc, F. et al. Human plasma protein N-glycosylation. Glycoconjugate J. 33, 1-35 (2015).
  6. Bladergroen, M. R. et al. Automation of high-throughput mass spectrometry-based plasma N-glycome analysis with linkage-specific sialic acid esterification. J. Proteome Res. (2015).