title: Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry.
abstract: An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar "glyco tune mix". When subjected to liquid chromatography-ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added13C-galactose led to absolute quantification in the same experiment. Graphical Abstract Discrepancy between desirable (grey bars) and real (green bars) relative ion abundance of equimolar amounts of glycans in positive mode ESI-MS.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Grünwald-Gruber
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thader
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dalik
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria. friedrich.altmann@boku.ac.at.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
mesh_terms: D002244:Carbon / Q000737:chemistry*; D002853:Chromatography, Liquid; D005690:Galactose / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry*; D012794:Sialic Acids / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D011134:Polysaccharides; D012794:Sialic Acids; D002244:Carbon; D005690:Galactose
keywords: Electrospray ionization; Mass spectrometry; N-Glycan; Quantitative glycomics; Sialylation