One filter, one sample, and the N- and O-glyco(proteo)me: toward a system to study disorders of protein glycosylation

Skeene, K., Walker, M. , Clarke, G., Bergström, E., Genever, P., Ungar, D. and Thomas-Oates, J. (2017) One filter, one sample, and the N- and O-glyco(proteo)me: toward a system to study disorders of protein glycosylation. Analytical Chemistry, 89(11), pp. 5840-5849. (doi:10.1021/acs.analchem.7b00143) (PMID:28453257)

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Abstract

A method has been developed for release/isolation of O-glycans from glycoproteins in whole cell lysates for mass spectrometric analysis. Cells are lysed in SDS, which is then exchanged for urea and ammonium bicarbonate in a centrifugal filter, before treating with NH4OH to release O-glycans. Following centrifugation, O-glycans are recovered in the filtrate. Sonication achieves O-glycan release in 1 h. Combining the established protocol for filter-aided N-glycan separation, here optimized for enhanced PNGase F efficiency, with the developed O-glycan release method allows analysis of both N- and O-glycans from one sample, in the same filter unit, from 0.5 to 1 million cells. The method is compatible with subsequent analysis of the residual protein by liquid chromatography-mass spectrometry (LC-MS) after glycan release. The medium throughput approach is amenable to analysis of biological replicates, offering a simple way to assess the often subtle changes to glycan profiles accompanying differentiation and disease progression, in a statistically robust way.

Item Type:Articles
Additional Information:K.S. gratefully acknowledges her Ph.D. funding from BristolMyers Squibb and the University of York teaching studentship. The York Centre of Excellence in Mass Spectrometry was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward, with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1; EP/M028127/1).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Walker, Mr Matthew
Authors: Skeene, K., Walker, M., Clarke, G., Bergström, E., Genever, P., Ungar, D., and Thomas-Oates, J.
College/School:College of Science and Engineering > School of Engineering > Biomedical Engineering
Journal Name:Analytical Chemistry
Publisher:American Chemical Society
ISSN:0003-2700
ISSN (Online):1520-6882
Published Online:28 April 2017
Copyright Holders:Copyright © 2017 American Chemical Society
First Published:First published in Analytical Chemistry 89(11): 5840-5849
Publisher Policy:Reproduced under a Creative Commons License

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