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
- 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)
- 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).
- 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)
- 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).
- 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).
- 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).
- Kammeijer, G. S. M. et al. Sialic acid linkage Analysis of Glycopeptides using CE-ESI-MS(/MS). Sci. Rep. 7, 3733 (2017).
- 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).
- Ramautar, R. et al. Enhancing the Coverage of the Urinary Metabolome by Sheathless Capillary Electrophoresis-Mass Spectrometry. Anal. Chem. 84 (2), 885–892 (2012).
- Huhn, C. et al. Relevance and use of capillary coatings in capillary electrophoresis–mass spectrometry. Anal. Bioanal. Chem. 396(1), 297-314 (2010).