Skeene, Kirsty, Walker, Matthew Joseph, Clarke, Graham et al. (4 more authors) (2017) One filter, one sample and the N- and O-glyco(proteo)me: towards a system to study disorders of protein glycosylation. : Toward a System to Study Disorders of Protein Glycosylation. Analytical Chemistry. pp. 5840-5849. ISSN 0003-2700
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.
Metadata
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Institution: | The University of York | ||||
Academic Units: | The University of York > Faculty of Sciences (York) > Chemistry (York) The University of York > Faculty of Sciences (York) > Biology (York) |
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Depositing User: | Pure (York) | ||||
Date Deposited: | 05 Jun 2017 12:00 | ||||
Last Modified: | 04 Jan 2024 00:14 | ||||
Published Version: | https://doi.org/10.1021/acs.analchem.7b00143 | ||||
Status: | Published | ||||
Refereed: | Yes | ||||
Identification Number: | https://doi.org/10.1021/acs.analchem.7b00143 | ||||
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Filename: ac7b00143_si_001.pdf
Description: Supporting information
Filename: Skeene_et_al_Anal_Chem_2017.pdf
Description: Skeene et al, Anal Chem 2017
Licence: CC-BY 2.5