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

TY - JOUR

T1 - One filter, one sample and the N- and O-glyco(proteo)me: towards a system to study disorders of protein glycosylation.

T2 - Toward a System to Study Disorders of Protein Glycosylation

AU - Skeene, Kirsty

AU - Walker, Matthew Joseph

AU - Clarke, Graham

AU - Bergström, Ed

AU - Genever, Paul

AU - Ungar, Daniel

AU - Thomas-Oates, Jane

PY - 2017/6/6

Y1 - 2017/6/6

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85020910281&partnerID=8YFLogxK

U2 - 10.1021/acs.analchem.7b00143

DO - 10.1021/acs.analchem.7b00143

M3 - Article

AN - SCOPUS:85020910281

SN - 0003-2700

VL - 89

SP - 5840

EP - 5849

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 11

ER -