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Glycoconj J. 2018 Jun 16. doi: 10.1007/s10719-018-9825-8. [Epub ahead of print]

Towards automation of glycomic profiling of complex biological materials.

Author information

1
Ludger Ltd, Culham Science Centre, Abingdon, OX14 3EB, United Kingdom. archana.shubhakar@ludger.com.
2
Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands. archana.shubhakar@ludger.com.
3
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.
4
Ludger Ltd, Culham Science Centre, Abingdon, OX14 3EB, United Kingdom.
5
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK. s.haslam@imperial.ac.uk.

Abstract

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.

KEYWORDS:

Automation; Glycan characterisation; MALDI-TOF-MS; Mouse tissues; N- and O-glycosylation; Permethylation

PMID:
29909447
DOI:
10.1007/s10719-018-9825-8

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