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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

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JournalAnalytical Chemistry
DateAccepted/In press - 28 Apr 2017
DateE-pub ahead of print - 28 Apr 2017
DatePublished (current) - 6 Jun 2017
Issue number11
Volume89
Number of pages10
Pages (from-to)5840-5849
Early online date28/04/17
Original languageEnglish

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.

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