High-Throughput Analysis of the Plasma N-Glycome by UHPLC

  • Barbara Adamczyk
  • Henning Stöckmann
  • Róisín O’Flaherty
  • Niclas G. Karlsson
  • Pauline M. RuddEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1503)


The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation.

Key words

N-linked glycosylation Plasma Glycan analysis Ultrahigh-performance liquid chromatography Robotics Automation High-throughput 



The authors acknowledge support from the European Union FP7 GastricGlycoExplorer ITN under grant agreement no. 316929. and HighGlycan under grant agreement no. 278535. We would like to acknowledge Dr. Karol Polom, Dr. Giovanni Corso, and Dr. Franco Roviello from University Hospital of Siena for providing gastric cancer serum samples. The UHPLC instrument was obtained with a grant from the IngaBritt and Arne Lundberg’s Research Foundation.


  1. 1.
    Merry AH, Merry CL (2005) Glycoscience finally comes of age. EMBO Rep 6(10):900–903CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3(2):97–130CrossRefPubMedGoogle Scholar
  3. 3.
    Lowe JB (2001) Glycosylation, immunity, and autoimmunity. Cell 104(6):809–812CrossRefPubMedGoogle Scholar
  4. 4.
    Lauc G et al (2016) Mechanisms of disease: the human N-glycome. Biochim Biophys Acta 1860:1574–1582CrossRefPubMedGoogle Scholar
  5. 5.
    Fuster MM, Esko JD (2005) The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 5(7):526–542CrossRefPubMedGoogle Scholar
  6. 6.
    Freeze HH, Aebi M (2005) Altered glycan structures: the molecular basis of congenital disorders of glycosylation. Curr Opin Struct Biol 15(5):490–498CrossRefPubMedGoogle Scholar
  7. 7.
    Szabo Z et al (2010) Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS. Electrophoresis 31(8):1389–1395CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Royle L et al (2008) HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal Biochem 376(1):1–12CrossRefPubMedGoogle Scholar
  9. 9.
    Ahn J et al (2010) Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 um sorbent. J Chromatogr B 878(34):403–408CrossRefGoogle Scholar
  10. 10.
    Marino K et al (2010) A systematic approach to protein glycosylation analysis: a path through the maze. Nat Chem Biol 6(10):713–723CrossRefPubMedGoogle Scholar
  11. 11.
    North SJ et al (2009) Mass spectrometry in the analysis of N-linked and O-linked glycans. Curr Opin Struct Biol 19(5):498–506CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Harvey DJ (2005) Proteomic analysis of glycosylation: structural determination of N-and O-linked glycans by mass spectrometry. Expert Rev Proteomics 2(1):87–101CrossRefPubMedGoogle Scholar
  13. 13.
    Wuhrer M, Deelder AM, Hokke CH (2005) Protein glycosylation analysis by liquid chromatography–mass spectrometry. J Chromatogr B 825(2):124–133CrossRefGoogle Scholar
  14. 14.
    Guile GR et al (1996) A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal Biochem 240(2):210–226CrossRefPubMedGoogle Scholar
  15. 15.
    Tharmalingam T et al (2013) Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides. Glycoconj J 30(2):137–146CrossRefPubMedGoogle Scholar
  16. 16.
    Royle L et al (2006) Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions. In: Brockhausen I (ed) Methods in molecular biology. Humana Press Inc, Totowa, NJ, pp 125–143Google Scholar
  17. 17.
    Knezevic A et al (2008) Variability, heritability and environmental determinants of human plasma N-glycome. J Proteome Res 8(2):694–701CrossRefGoogle Scholar
  18. 18.
    Arnold JN et al (2011) Novel glycan biomarkers for the detection of lung cancer. J Proteome Res 10(4):1755–1764CrossRefPubMedGoogle Scholar
  19. 19.
    Reusch D et al (2013) High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples. Anal Biochem 432(2):82–89CrossRefPubMedGoogle Scholar
  20. 20.
    Trbojević Akmačić I et al (2015) High-throughput glycomics: optimization of sample preparation. Biochemistry (Mosc) 80(7):934–942CrossRefGoogle Scholar
  21. 21.
    Stöckmann H et al (2013) Automated, high-throughput IgG-antibody glycoprofiling platform. Anal Chem 85(18):8841–8849CrossRefPubMedGoogle Scholar
  22. 22.
    Shubhakar A et al (2015) High-throughput analysis and automation for glycomics studies. Chromatographia 78(5–6):321–333CrossRefPubMedGoogle Scholar
  23. 23.
    Ruhaak LR et al (2008) Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins. Anal Chem 80(15):6119–6126CrossRefPubMedGoogle Scholar
  24. 24.
    Stöckmann H et al (2015) Automated, high-throughput serum glycoprofiling platform. Integr Biol 7(9):1026–1032CrossRefGoogle Scholar
  25. 25.
    Campbell MP, Royle L, Rudd PM (2015) GlycoBase and autoGU: resources for interpreting HPLC-Glycan data. Methods Mol Biol 1273:17–28CrossRefPubMedGoogle Scholar
  26. 26.
    Adamczyk B et al (2012) Characterization of fibrinogen glycosylation and its importance for serum/plasma N-glycome analysis. J Proteome Res 12(1):444–454CrossRefPubMedGoogle Scholar
  27. 27.
    Stöckmann H et al (2015) Ultrahigh throughput, ultrafiltration-based N-Glycomics platform for ultraperformance liquid chromatography (ULTRA3). Anal Chem 87(16):8316–8322CrossRefPubMedGoogle Scholar
  28. 28.
    Harvey DJ et al (2009) Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds. Proteomics 9(15):3796–3801CrossRefPubMedGoogle Scholar
  29. 29.
    Saldova R et al (2014) Association of N-glycosylation with breast carcinoma and systemic features using high-resolution quantitative UPLC. J Proteome Res 13(5):2314–2327CrossRefPubMedGoogle Scholar
  30. 30.
    Bones J et al (2010) Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from host defense response mechanisms. J Proteome Res 10(3):1246–1265CrossRefGoogle Scholar
  31. 31.
    Bones J et al (2010) Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation. Anal Chem 82(24):10208–10215CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Barbara Adamczyk
    • 1
    • 2
  • Henning Stöckmann
    • 2
    • 3
  • Róisín O’Flaherty
    • 2
  • Niclas G. Karlsson
    • 1
  • Pauline M. Rudd
    • 2
    Email author
  1. 1.Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska AcademyUniversity of GothenburgGothenburgSweden
  2. 2.NIBRT GlycoScience Group, NIBRT–The National Institute for Bioprocessing Research and TrainingDublinIreland
  3. 3.AbbVie Inc.North ChicagoUSA

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