title: Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.
abstract: Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
authors.forename: Shiyue
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
authors.forename: Nadia
authors.initials: N
authors.lastname: Tello
authors.affiliation: GlycoScientific, Athens, GA, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Harvey
authors.affiliation: GlycoScientific, Athens, GA, USA.
authors.forename: Barry
authors.initials: B
authors.lastname: Boyes
authors.affiliation: GlycoScientific, Athens, GA, USA.
authors.forename: Ron
authors.initials: R
authors.lastname: Orlando
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
authors.forename: Yehia
authors.initials: Y
authors.lastname: Mechref
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D054794:Glycomics / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000032:analysis*; D012015:Reference Standards
chemical_list: D011134:Polysaccharides
keywords: Glycomics; Internal standard; LC-MS/MS; Permethylation; iGlycoMAb