One Filter, One Sample, and the N- and O-Glyco(proteo)me: Toward a System to Study Disorders of Protein Glycosylation.

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

doi:10.1021/acs.analchem.7b00143

Kirsty Skeene, Jane Thomas-Oates + Show 5 more

Open Access

https://doi.org/10.1021/acs.analchem.7b00143

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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.

Highlights

(ER) and the Golgi apparatus) and is dependent on their expression levels, location, and activity
The method makes use of a centrifugal filter as a simple reaction vessel in which glycoproteins can be treated with NH4OH solution to release O-glycans by β-elimination
The method can be conveniently combined with the established FANGS protocol, to release, isolate, and analyze both N- and O-glycans using one sample, in the same filter unit, enabling analysis of the whole glycome

Summary

Introduction

(ER) and the Golgi apparatus) and is dependent on their expression levels, location, and activity. It is clear that enzymes are sorted using transport vesicles, which are targeted to their appropriate locations during the vesicle tethering process This is mediated by the conserved oligomeric Golgi (COG) protein complex, which has been shown to play a central role in maintaining the fidelity of glycosylation in all eukaryotic cells.[28−31]. Since some genetic diseases can occur as a result of a genetic defect that affects N- and O-glycosylation simultaneously, whereas some defects only affect one glycan type, analysis of both N- and O- Such a one-sample-one-pot method has the advantage of reducing the amount of material needed to carry out such studies and offers the potential, after N- and O-glycan release, to analyze the remaining protein, enabling a complementary proteomic comparison between samples. Still needed to release and isolate O-glycans from cultured cells for mass spectrometric analysis

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