Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.
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This work was supported by a Business Innovation Voucher funded by IBCarb and Biotechnology and Biological Sciences Research Council (BBSRC) and by the European Union Seventh Framework Programmes HighGlycan (Grant Number 278535). Support was also provided by grants BB/K016164/1 and BB/F008309/1 from the Biotechnology and Biological Sciences Research Council. The authors are also grateful to Manfred Wuhrer for critical reading of this manuscript.
Conflicts of interest
The authors declare the following competing financial interest(s): Ludger Ltd. is a commercial bioscience company specializing in development and validation of glycoprofiling technology. The following authors, A.S., D.I.R.S. and D.L.F. are employed by Ludger Ltd. Products from Ludger were used in this research.
All applicable international, national, and institutional guidelines for the care and use of animals were followed.
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Shubhakar, A., Pang, P., Fernandes, D.L. et al. Towards automation of glycomic profiling of complex biological materials. Glycoconj J 35, 311–321 (2018). https://doi.org/10.1007/s10719-018-9825-8
- N- and O-glycosylation
- Mouse tissues
- Glycan characterisation