1 research outputs found
Mass Spectrometric Quantification of N‑Linked Glycans by Reference to Exogenous Standards
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