Filter-Aided N-Glycan Separation (FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling

TY - JOUR

T1 - Filter-Aided N-Glycan Separation (FANGS):

T2 - A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling

AU - Abdul Rahman, Salina

AU - Bergström, Ed

AU - Watson, Christopher J.

AU - Wilson, Katherine M.

AU - Ashford, David A.

AU - Thomas, Jerry R.

AU - Ungar, Daniel

AU - Thomas-Oates, Jane E.

PY - 2014

Y1 - 2014

N2 - We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method "filter-aided N-glycan separation" and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples. © 2014 American Chemical Society.

AB - We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method "filter-aided N-glycan separation" and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples. © 2014 American Chemical Society.

KW - cultured mammalian cells

KW - filter-aided sample preparation

KW - MALDI-MS

KW - N -glycans

KW - permethylation

KW - SDS solubilization

UR - http://www.scopus.com/inward/record.url?scp=84896747872&partnerID=8YFLogxK

U2 - 10.1021/pr401043r

DO - 10.1021/pr401043r

M3 - Article

C2 - 24450425

SN - 1535-3893

VL - 13

SP - 1167

EP - 1176

JO - Journal of Proteome Research

JF - Journal of Proteome Research

IS - 3

ER -