A Rapid Array-Based Approach to N-Glycan Profiling of Cultured Cells

J Proteome Res. 2019 Oct 4;18(10):3630-3639. doi: 10.1021/acs.jproteome.9b00303. Epub 2019 Sep 19.

Abstract

Typically, N-glycosylation studies done on cultured cells require up to millions of cells followed by lengthy preparation to release, isolate, and profile N-glycans. To overcome these limitations, we report a rapid array-based workflow for profiling N-glycan signatures from cells, adapted from imaging mass spectrometry used for on-tissue N-glycan profiling. Using this approach, N-glycan profiles from a low-density array of eight cell chambers could be reported within 4 h of completing cell culture. Approaches are demonstrated that account for background N-glycans due to serum media. Normalization procedures are shown. The method is robust and reproducible, requiring as few as 3000 cells per replicate with a 3-20% coefficient of variation to capture label-free profiles of N-glycans. Quantification by stable isotopic labeling of N-glycans in cell culture is demonstrated and adds no additional time to preparation. Utility of the method is demonstrated by measurement of N-glycan turnover rates due to induction of oxidative stress in human primary aortic endothelial cells. The developed method and ancillary tools serve as a foundational launching point for rapid profiling of N-glycans ranging from high-density arrays down to single cells in culture.

Keywords: N-glycan; N-glycosylation; array; imaging mass spectrometry; label-free; single cell; stable isotope.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aorta / cytology
  • Aorta / metabolism
  • Endothelial Cells / chemistry
  • Endothelial Cells / metabolism
  • Glycomics / methods*
  • Humans
  • Isotope Labeling / methods
  • Mass Spectrometry / methods
  • Methods
  • Oxidative Stress
  • Polysaccharides / analysis*

Substances

  • Polysaccharides