Faculty Collaboration Database

Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards. J Proteome Res 2016 09 02;15(9):2969-80

Date

07/20/2016

Pubmed ID

27432553

Pubmed Central ID

PMC5501980

DOI

10.1021/acs.jproteome.6b00132

Scopus ID

2-s2.0-84985993219   29 Citations

Abstract

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

Author List

Mehta N, Porterfield M, Struwe WB, Heiss C, Azadi P, Rudd PM, Tiemeyer M, Aoki K

Author

Kazuhiro Aoki PhD Associate Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Humans
Oligosaccharides
Polysaccharides
Proteomics
Reference Standards
Reproducibility of Results
Sensitivity and Specificity
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Staining and Labeling