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Methods Mol Biol. 2017;1503:185-196.

High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells.

Author information

1
Center for Proteomics and Metabolomics, Leiden University Medical Center, Postzone S3, Postbus 9600, 2300, RC, Leiden, The Netherlands. s.holst@lumc.nl.
2
Department of Surgery, Leiden University Medical Center, Postbus 9600, RC, 2300, Leiden, The Netherlands.
3
Department of Molecular Cell Biology and Immunology, VU University Medical Centre, Amsterdam, The Netherlands.
4
Center for Proteomics and Metabolomics, Leiden University Medical Center, Postzone S3, Postbus 9600, 2300, RC, Leiden, The Netherlands.
5
University Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59 000, Lille, France.
6
Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, Amsterdam, 1081, HV, The Netherlands.
7
Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, Leiden, 2333, ZA, The Netherlands.

Abstract

The current protocols for glycomic analysis of cells often require a large quantity of material (5-20 million cells). In order to analyze the N-glycosylation from small amounts of cells (≤1 million) as obtained from, for example, primary cell lines or cell sorting, and in a higher throughput approach, we set up a robust 96-well format PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid stabilization, cleanup, and MALDI-TOF-MS analysis. We further evaluated the influence of PNGase F incubation time on the N-glycan profile.

KEYWORDS:

Cells; Ethyl esterification; MALDI-TOF-MS; N-glycosylation; PNGase F digestion; PVDF filter plates

PMID:
27743367
DOI:
10.1007/978-1-4939-6493-2_14
[Indexed for MEDLINE]

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