Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling.
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.
Department of Chemistry, ‡Centre of Excellence in Mass Spectrometry, and §Department of Biology, University of York , York YO10 5DD, United Kingdom.
Salina
S
Abdul Rahman
Ed
E
Bergström
Christopher J
CJ
Watson
Katherine M
KM
Wilson
David A
DA
Ashford
Jerry R
JR
Thomas
Daniel
D
Ungar
Jane E
JE
Thomas-Oates
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002457:Cell Extracts / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D003412:Cricetulus; D005374:Filtration; D006023:Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002457:Cell Extracts; D006023:Glycoproteins; D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase