Liquid chromatography-mass spectrometry

We develop liquid chromatography-mass spectrometry approaches for protein glycosylation analysis – predominantly at the level of glycopeptide analysis – and apply them for both in-depth glycosylation analysis and glycosylation profiling.

With respect to in-depth characterization, we have recently optimized collisional fragmentation conditions for glycopeptide analysis by ESI-QTOF-MS, in collaboration with Bruker Daltonics and the group of Dr. Daniel Kolarich.1 Likewise, we developed a workflow integrating C18-reverse phase and graphitized carbon LC-MS of glycopeptides for improved coverage of glycosylation sites.2

With respect to glycosylation profiling by LC-MS, we have established streamlined workflows for the glycosylation analysis of immunoglobulins as well as other plasma glycoproteins. The workflows include high-throughput sample preparation, rapid high-sensitivity nano-scale LC-MS detection, and largely automated data processing using our software suite LaCyTools.3,4

Capillary electrophoresis–electrospray ionization-mass spectrometry

Glycosylation, being one of the most heterogeneous post translation modifications, needs sensitive analytical platforms. Capillary electrophoresis (CE) hyphenated to a quadruple time-of-flight mass spectrometer (MS) with electrospray ionization (ESI) is such a platform, especially using a sheathless interface were analytes can be measured down to 0.2 amol when using a dopant enriched nitrogen gas.5 The consumption of minimal sample amounts, compared to other techniques, makes the platform especially attractive for precious samples. Its sensitivity is particularly suited for in-depth characterization on the glycopeptide level.


Two recent studies show the relevance of using such a system for the analysis of glycopeptides compared to nanoLC–ESI-MS.5,6 However this set-up is not only used for glyco(proteo)mics,7,8 such as labelled glycans, glycopeptides and glycoprotein analysis, but also for metabolic studies9 or the analysis of other PTMs.10

  1. Hinneburg, H. et al. The art of destruction: Optimizing collision energies in quadropole-time of flight (q-Tof) instruments for glycopeptide-based glycoproteomics. J. Am. Soc. Mass. Spectrom. 27, 507-519 (2016)
  2. Stavenhagen, K., Plomp, R. & Wuhrer, M. Site-Specific Protein N- and O-Glycosylation Analysis by a C18-Porous Graphitized Carbon-Liquid Chromatography-Electrospray Ionization Mass Spectrometry Approach Using Pronase Treated Glycopeptides. Anal. Chem. 87, 11691-11699 (2015).
  3. Falck, D., Jansen, B. C., de Haan, N. & Wuhrer, M. High-throughput analysis of IgG Fc glycopeptides by LC-MS. Methods Mol. Biol. 1503, 31-47 (2017)
  4. Jansen, B. C. et al. LaCyTools: A Targeted Liquid Chromatography-Mass Spectrometry Data Processing Package for Relative Quantitation of Glycopeptides. J. Proteome Res. 15, 2198-2210 (2016).
  5. Kammeijer, G. S. M. et al. Dopant Enriched Nitrogen Gas Combined with Sheathless Capillary Electrophoresis–Electrospray Ionization-Mass Spectrometry for Improved Sensitivity and Repeatability in Glycopeptide Analysis. Anal. Chem. 88, 5849-5856 (2016).
  6. Heemskerk, A. A. et al. Ultra-Low Flow Electrospray Ionization-Mass Spectrometry for Improved Ionization Efficiency in Phosphoproteomics. Anal. Chem. 84 (10), 4552–4559 (2012).
  7. Kammeijer, G. S. M. et al. Sialic acid linkage Analysis of Glycopeptides using CE-ESI-MS(/MS). Sci. Rep. 7, 3733 (2017).
  8. Heemskerk, A. A. et al. Coupling porous sheathless interface MS with transient-ITP in neutral capillaries for improved sensitivity in glycopeptide analysis. Electrophoresis 34, 383-387 (2013).
  9. Ramautar, R. et al. Enhancing the Coverage of the Urinary Metabolome by Sheathless Capillary Electrophoresis-Mass Spectrometry. Anal. Chem. 84 (2), 885–892 (2012).
  10. Huhn, C. et al. Relevance and use of capillary coatings in capillary electrophoresis–mass spectrometry. Anal. Bioanal. Chem. 396(1), 297-314 (2010).