title: Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.
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.
authors.forename: Nickita
authors.initials: N
authors.lastname: Mehta
authors.forename: Mindy
authors.initials: M
authors.lastname: Porterfield
authors.affiliation: National Institute for Bioprocessing Research and Training (NIBRT) , Dublin, Ireland.
authors.forename: Weston B
authors.initials: WB
authors.lastname: Struwe
authors.forename: Christian
authors.initials: C
authors.lastname: Heiss
authors.forename: Parastoo
authors.initials: P
authors.lastname: Azadi
authors.affiliation: National Institute for Bioprocessing Research and Training (NIBRT) , Dublin, Ireland.|University College , Dublin, Ireland.
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.forename: Michael
authors.initials: M
authors.lastname: Tiemeyer
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoki
mesh_terms: D000818:Animals; D006801:Humans; D009844:Oligosaccharides / Q000592:standards; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000592:standards; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000592:standards; D013194:Staining and Labeling
chemical_list: D009844:Oligosaccharides; D011134:Polysaccharides
keywords: N-glycan; mass spectrometry; permethylation; quantification; standard