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

par Emmanuel Maes - publié le , mis à jour le

Voici un article qui nous est recommandé à la lecture. Analyses rapides de O- et de N-Glycanes.
Kirsty Skeene, Matthew Walker, Graham Clarke, Ed Bergström, Paul Genever, Daniel Ungar and Jane Thomas-Oates

Bonne lecture

"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 Oglycans.
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."

Skeene K. et al, Anal. Chem, 2017, 89, 5840-5849