title: The use of a xylosylated plant glycoprotein as an internal standard accounting for N-linked glycan cleavage and sample preparation variability.
abstract: BACKGROUND
  Traditionally, free oligosaccharide internal standards are used to account for variability in glycan relative quantification experiments by mass spectrometry. However, a more suitable internal standard would be a glycoprotein, which could also control for enzymatic cleavage efficiency, allowing for more accurate quantitative experiments.
  
  
  METHODS
  Hydrophobic, hydrazide N-linked glycan reagents (both native and stable-isotope labeled) are used to derivatize and differentially label N-linked glycan samples for relative quantification, and the samples are analyzed by a reversed-phase liquid chromatography chip system coupled online to a Q-Exactive mass spectrometer. The inclusion of two internal standards, maltoheptaose (previously used) and horseradish peroxidase (HRP) (novel), is studied to demonstrate the effectiveness of using a glycoprotein as an internal standard in glycan relative quantification experiments.
  
  
  RESULTS
  HRP is a glycoprotein containing a xylosylated N-linked glycan, which is unique from mammalian N-linked glycans. Thus, the internal standard xylosylated glycan could be detected without interference to the sample. Additionally, it was shown that differences in cleavage efficiency can be detected by monitoring the HRP glycan. In a sample where cleavage efficiency variation is minimal, the HRP glycan performs as well as maltoheptaose.
  
  
  CONCLUSIONS
  Because the HRP glycan performs as well as maltoheptaose but is also capable of correcting and accounting for cleavage variability, it is a more versatile internal standard and will be used in all subsequent biological studies. Because of the possible lot-to-lot variation of an enzyme, differences in biological matrix, and variable enzyme activity over time, it is a necessity to account for glycan cleavage variability in glycan relative quantification experiments.
authors.affiliation: W.M. Keck Fourier Transform Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: S Hunter
authors.initials: SH
authors.lastname: Walker
authors.forename: Amber D
authors.initials: AD
authors.lastname: Taylor
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000592:standards*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000592:standards*; D010940:Plant Proteins / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D012015:Reference Standards
chemical_list: D006023:Glycoproteins; D010940:Plant Proteins; D011134:Polysaccharides