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

Kirsty Skeene, Matthew Joseph Walker, Graham Clarke, Ed Bergström, Paul Genever, Daniel Ungar*, Jane Thomas-Oates

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)5840-5849
Number of pages10
JournalAnalytical Chemistry
Issue number11
Early online date28 Apr 2017
Publication statusPublished - 6 Jun 2017

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