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

Methods Mol Biol. 2017;1503:185-196. doi: 10.1007/978-1-4939-6493-2_14.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Chromatography, Liquid / methods
  • Esterification
  • Glycomics / methods*
  • Glycosylation
  • High-Throughput Screening Assays / methods
  • Humans
  • N-Acetylneuraminic Acid / chemistry
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Polysaccharides / analysis*
  • Polysaccharides / isolation & purification
  • Polysaccharides / metabolism
  • Solid Phase Extraction / methods
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*

Substances

  • Polysaccharides
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • N-Acetylneuraminic Acid