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dc.contributor.authorHinneburg, Hannes
dc.contributor.authorChatterjee, Sayantani
dc.contributor.authorSchirmeister, Falko
dc.contributor.authorNguyen-Khuong, Terry
dc.contributor.authorPacker, Nicolle H
dc.contributor.authorRapp, Erdmann
dc.contributor.authorThaysen-Andersen, Morten
dc.description.abstractDeep 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.en_US
dc.publisherAmerican Chemical Society (ACS Publications)en_US
dc.relation.ispartofjournalAnalytical Chemistryen_US
dc.subject.fieldofresearchAnalytical Chemistryen_US
dc.subject.fieldofresearchOther Chemical Sciencesen_US
dc.subject.keywordsScience & Technologyen_US
dc.subject.keywordsPhysical Sciencesen_US
dc.subject.keywordsRELEASED N-GLYCANSen_US
dc.titlePost-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomicsen_US
dc.typeJournal articleen_US
dc.type.descriptionC1 - Articlesen_US
dcterms.bibliographicCitationHinneburg, H; Chatterjee, S; Schirmeister, F; Nguyen-Khuong, T; Packer, NH; Rapp, E; Thaysen-Andersen, M, Post-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics, Analytical Chemistry, 2019, 91 (7), pp. 4559-4567en_US
gro.hasfulltextNo Full Text
gro.griffith.authorPacker, Nicki

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