期刊
JOURNAL OF PROTEOME RESEARCH
卷 13, 期 3, 页码 1167-1176出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr401043r
关键词
cultured mammalian cells; SDS solubilization; N-glycans; filter-aided sample preparation; MALDI-MS; permethylation
资金
- Ministry of Health, Malaysia
- Yorkshire Forward
- Northern Way Initiative
- Marie Curie IRG [201098]
- Biotechnology and Biological Sciences Research Council [BB/F006993/1] Funding Source: researchfish
- BBSRC [BB/F006993/1] Funding Source: UKRI
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.
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