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  • Post-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics

    Author(s)
    Hinneburg, Hannes
    Chatterjee, Sayantani
    Schirmeister, Falko
    Nguyen-Khuong, Terry
    Packer, Nicolle H
    Rapp, Erdmann
    Thaysen-Andersen, Morten
    Griffith University Author(s)
    Packer, Nicki
    Year published
    2019
    Metadata
    Show full item record
    Abstract
    Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to ...
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    Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc. 2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.
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    Journal Title
    Analytical Chemistry
    Volume
    91
    Issue
    7
    DOI
    https://doi.org/10.1021/acs.analchem.8b05720
    Subject
    Analytical chemistry
    Other chemical sciences
    Science & Technology
    Physical Sciences
    Chemistry
    RELEASED N-GLYCANS
    Publication URI
    http://hdl.handle.net/10072/404974
    Collection
    • Journal articles

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