title: Twoplex12/13C6aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.
abstract: Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MSEworkflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral12C6and13C6aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex12C6/13C6aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation.
authors.affiliation: Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Dublin, Ireland.
authors.forename: Simone
authors.initials: S
authors.lastname: Albrecht
authors.affiliation: Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Dublin, Ireland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mittermayr
authors.affiliation: Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Dublin, Ireland.
authors.forename: Josh
authors.initials: J
authors.lastname: Smith
authors.affiliation: Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Dublin, Ireland.
authors.forename: Silvia Millán
authors.initials: SM
authors.lastname: Martín
authors.affiliation: Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Dublin, Ireland.|Department of Life Sciences, School of Science, Institute of Technology Sligo, Sligo, Ireland.
authors.forename: Margaret
authors.initials: M
authors.lastname: Doherty
authors.affiliation: Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Dublin, Ireland.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bones
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D054794:Glycomics / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
chemical_list: D000814:Aniline Compounds; D019158:N-Acetylneuraminic Acid; C023650:aniline
keywords: DMT-MM; Glycan quantitation; Glycoproteomics; LC-MS; Sialic acid; Stable isotope aniline