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

Stephanie Holst; Gabi W van Pelt; Wilma E Mesker; Rob A Tollenaar; Ana I Belo; Irma van Die; Yoann Rombouts; Manfred Wuhrer

doi:10.1007/978-1-4939-6493-2_14

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

Stephanie Holst, Gabi W van Pelt, Wilma E Mesker, Rob A Tollenaar, Ana I Belo, Irma van Die, Yoann Rombouts, Manfred Wuhrer

Molecular cell biology and Immunology

Research output: Contribution to journal › Article › Academic › peer-review

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.

Original language	English
Pages (from-to)	185-196
Number of pages	12
Journal	Methods in Molecular Biology
Volume	1503
DOIs	https://doi.org/10.1007/978-1-4939-6493-2_14
Publication status	Published - 2017

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10.1007/978-1-4939-6493-2_14

Cite this

Holst, S., van Pelt, G. W., Mesker, W. E., Tollenaar, R. A., Belo, A. I., van Die, I., ... Wuhrer, M. (2017). High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells. Methods in Molecular Biology, 1503, 185-196. https://doi.org/10.1007/978-1-4939-6493-2_14

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title = "High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells",

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 = "Animals, Cell Line, Tumor, Chromatography, Liquid, Esterification, Glycomics, Glycosylation, High-Throughput Screening Assays, Humans, N-Acetylneuraminic Acid, Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase, Polysaccharides, Solid Phase Extraction, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Journal Article",

author = "Stephanie Holst and {van Pelt}, {Gabi W} and Mesker, {Wilma E} and Tollenaar, {Rob A} and Belo, {Ana I} and {van Die}, Irma and Yoann Rombouts and Manfred Wuhrer",

year = "2017",

doi = "10.1007/978-1-4939-6493-2_14",

language = "English",

volume = "1503",

pages = "185--196",

journal = "Methods in Molecular Biology",

issn = "1064-3745",

publisher = "Humana Press",

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High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells. / Holst, Stephanie; van Pelt, Gabi W; Mesker, Wilma E; Tollenaar, Rob A; Belo, Ana I; van Die, Irma; Rombouts, Yoann; Wuhrer, Manfred.

In: Methods in Molecular Biology, Vol. 1503, 2017, p. 185-196.

Research output: Contribution to journal › Article › Academic › peer-review

TY - JOUR

T1 - High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells

AU - Holst, Stephanie

AU - van Pelt, Gabi W

AU - Mesker, Wilma E

AU - Tollenaar, Rob A

AU - Belo, Ana I

AU - van Die, Irma

AU - Rombouts, Yoann

AU - Wuhrer, Manfred

PY - 2017

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N2 - 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.

AB - 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.

KW - Animals

KW - Cell Line, Tumor

KW - Chromatography, Liquid

KW - Esterification

KW - Glycomics

KW - Glycosylation

KW - High-Throughput Screening Assays

KW - Humans

KW - N-Acetylneuraminic Acid

KW - Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase

KW - Polysaccharides

KW - Solid Phase Extraction

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Journal Article

U2 - 10.1007/978-1-4939-6493-2_14

DO - 10.1007/978-1-4939-6493-2_14

M3 - Article

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VL - 1503

SP - 185

EP - 196

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -