Glycosylation is one of the most abundant and important protein modifications and it is associated with a multitude of diseases.1,2 Next to this, glycosylation plays an important role in the efficacy of biopharmaceuticals.3 However, the analysis of glycans is a major challenge due to its heterogeneous character and low abundance in complex mixtures. Recently, a 96-well based high-throughput polyvinylidene difluoride (PVDF) membrane-based approach was developed for the analysis of released N-glycans from cells.4 Here, we further developed the PVDF membrane approach to enhance its sensitivity and to allow the analysis of N-glycans in low abundant samples. In addition, to explore the efficiency of the release on the PVDF membrane as well as the sensitivity of this approach, immunoglobulin G (IgG) was used as a standard.
To optimize the release of the N-glycans, several steps in the PVDF membrane approach were evaluated, such as protein immobilization, membrane blocking and N-glycan release conditions. Different concentrations of IgG (2- 20 µg) were applied and immobilized to the PVDF-membrane in 96-well plate format. Protein denaturation was performed using 8M guanidine HCl and 100mM dithiothreitol (DTT). Nonidet P-40 (NP-40) and polyvinylpyrrolidone (PVP40) at different concentrations were studied to block the membrane prior to the N-glycans release by PNGase F. After hydrophilic interaction chromatography based solid phase extraction (HILIC-SPE) purification, the resulting N-glycans were directly analyzed by matrix-assisted laser desorption-ionization – time of flight mass spectrometry (MALDI-TOF-MS) and by using an internal standard, N-glycans could be quantified.
The optimized membrane-based protocol showed the potential for high sensitivity analysis. Blocking with NP-40 resulted in a more efficient release, as compared to PVP40. For instance, the relative intensity of N-glycans were enhanced, and more N-glycan compositions were detected using NP-40. To further evaluate and improve the sensitivity of this method, future research will focus on using starting amounts of protein in the ng range. Furthermore, the released N-glycans will be analyzed by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). The potential advantage of CE-ESI-MS is that the additional purification step (HILIC-SPE) can be avoided, and the overall higher sensitivity of the method. However, for the analysis with CE-ESI-MS a charge should be introduced, therefore, chemical labelling procedures at the reducing end of the N-glycans by different labels (e.g. the hydrazide Girard’s reagent P (GirP)) are currently under investigation.
- Rombouts, Y.; Ewing, E.; Van de Stadt, L. A.; Selman, M. H.; Trouw, L. A.; Deelder, A. M.; Huizinga, T. W.; Wuhrer, M.; van Schaardenburg, D.; Toes, R. E.; Scherer, H. U. Ann Rheum Dis 2015, 74, 234-241.
- Varki, A.; .Glycobiology, 2017,27(1): 3 -49.
- Walsh,G.; Nat.Biotechnol., 2014, pp. 992-1000.
- Holst, S.; van Pelt, G.W.; Mesker, W.E.; Tollenaar R.A.; Belo A.I.; Van Die I.; Rombouts, Y.; Wuhrer, M.; Methods in Molecular Biology, volume 1503.