Site-Specific N- and O-Glycopeptide Analysis Using an Integrated C18-PGC-LC-ESI-QTOF-MS/MS Approach

  • Kathrin Stavenhagen
  • Hannes Hinneburg
  • Daniel Kolarich
  • Manfred WuhrerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1503)


The vast heterogeneity of protein glycosylation, even of a single glycoprotein with only one glycosylation site, can give rise to a set of macromolecules with different physicochemical properties. Thus, the use of orthogonal approaches for comprehensive characterization of glycoproteins is a key requirement. This chapter describes a universal workflow for site-specific N- and O-glycopeptide analysis. In a first step glycoproteins are treated with Pronase to generate glycopeptides containing small peptide sequences for enhanced glycosylation site assignment and characterization. These glycopeptides are then separated and detected using an integrated C18-porous graphitized carbon-liquid chromatography (PGC-LC) setup online coupled to a high-resolution electrospray ionization (ESI)-quadrupole time-of-flight (QTOF)-mass spectrometer operated in a combined higher- and lower-energy CID (stepping-energy CID) mode. The LC-setup allows retention of more hydrophobic glycopeptides on C18 followed by subsequent capturing of C18-unbound (glyco)peptides by a downstream placed PGC stationary phase. Glycopeptides eluted from both columns are then analyzed within a single analysis in a combined data acquisition mode. Stepping-energy CID results in B- and Y-ion fragments originating from the glycan moiety as well as b- and y-ions derived from the peptide part. This allows simultaneous site-specific identification of the glycan and peptide sequence of a glycoprotein.

Key words

Glycoproteomics N-glycopeptide O-glycopeptide Mass spectrometry Porous graphitized carbon QTOF-MS Stepping-energy CID C18-PGC-LC 


  1. 1.
    Stavenhagen K, Hinneburg H, Thaysen-Andersen M et al (2013) Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. J Mass Spectrom 48:627–639CrossRefPubMedGoogle Scholar
  2. 2.
    Thaysen-Andersen M, Packer NH (2014) Advances in LC-MS/MS-based glycoproteomics: getting closer to system-wide site-specific mapping of the N- and O-glycoproteomes. Biochim Biophys Acta 1844:1437–1452CrossRefPubMedGoogle Scholar
  3. 3.
    Nilsson J, Halim A, Grahn A, Larson G (2013) Targeting the glycoproteome. Glycoconj J 30:119–136CrossRefPubMedGoogle Scholar
  4. 4.
    Alley WR, Mann BF, Novotny MV (2013) High-sensitivity analytical approaches for the structural characterization of glycoproteins. Chem Rev 113:2668–2732CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Desaire H (2013) Glycopeptide analysis, recent developments and applications. Mol Cell Proteomics 12:893–901CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Zauner G, Koeleman CAM, Deelder AM, Wuhrer M (2010) Protein glycosylation analysis by HILIC-LC-MS of Proteinase K-generated N- and O-glycopeptides. J Sep Sci 33:903–910CrossRefPubMedGoogle Scholar
  7. 7.
    Hua S, Nwosu CC, Strum JS et al (2012) Site-specific protein glycosylation analysis with glycan isomer differentiation. Anal Bioanal Chem 403:1291–1302CrossRefPubMedGoogle Scholar
  8. 8.
    Nwosu CC, Huang J, Aldredge D et al (2013) In-gel nonspecific proteolysis for elucidating glycoproteins: a method for targeted protein-specific glycosylation analysis in complex protein mixtures. Anal Chem 85:956–963CrossRefPubMedGoogle Scholar
  9. 9.
    Larsen MR, Højrup P, Roepstorff P (2005) Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry. Mol Cell Proteomics 4:107–119CrossRefPubMedGoogle Scholar
  10. 10.
    Temporini C, Perani E, Calleri E et al (2007) Pronase-immobilized enzyme reactor: an approach for automation in glycoprotein analysis by LC/LC-ESI/MS pronase-immobilized enzyme reactor: an approach for automation in glycoprotein analysis by LC/LC-ESI/MSn. Anal Chem 79:355–363CrossRefPubMedGoogle Scholar
  11. 11.
    Lewandrowski U, Sickmann A (2010) Online dual gradient reversed-phase/porous graphitized carbon nanoHPLC for proteomic applications. Anal Chem 82:5391–5396CrossRefPubMedGoogle Scholar
  12. 12.
    Stavenhagen K, Plomp R, Wuhrer M (2015) Site-specific protein N- and O-glycosylation analysis by a C18-porous graphitized carbon-liquid chromatography-electrospray ionization mass spectrometry approach using pronase treated glycopeptides. Anal Chem 87:11691–11699CrossRefPubMedGoogle Scholar
  13. 13.
    Ruhaak LR, Deelder AM, Wuhrer M (2009) Oligosaccharide analysis by graphitized carbon liquid chromatography-mass spectrometry. Anal Bioanal Chem 394:163–174CrossRefPubMedGoogle Scholar
  14. 14.
    Jensen PH, Karlsson NG, Kolarich D, Packer NH (2012) Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc 7:1299–1310CrossRefPubMedGoogle Scholar
  15. 15.
    Stavenhagen K, Kolarich D, Wuhrer M (2014) Clinical glycomics employing graphitized carbon liquid chromatography–mass spectrometry. Chromatographia 78:307–320CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Packer NH, Lawson MA, Jardine DR, Redmond JW (1998) A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj J 15:737–747CrossRefPubMedGoogle Scholar
  17. 17.
    Thaysen-Andersen M, Wilkinson BL, Payne RJ, Packer NH (2011) Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS. Electrophoresis 32:3536–3545CrossRefPubMedGoogle Scholar
  18. 18.
    Alley W, Mechref Y, Novotny MV (2009) Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides. Rapid Commun Mass Spectrom 23:495–505CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Davies MJ, Smith KD, Harbin AM, Hounsell EF (1992) High-performance liquid chromatography of oligosaccharide alditols and glycopeptides on a graphitized carbon column. J Chromatogr 609:125–131CrossRefPubMedGoogle Scholar
  20. 20.
    Wagner-Rousset E, Bednarczyk A, Bussat M-C et al (2008) The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies. J Chromatogr B 872:23–37CrossRefGoogle Scholar
  21. 21.
    Hinneburg H, Stavenhagen K, Schweiger-Hufnagel U et al (2016) The art of destruction: optimizing collision energies in quadrupole-time of flight (Q-TOF) instruments for glycopeptide-based glycoproteomics. J Am Soc Mass Spectrom 27:507–519. doi: 10.1007/s13361-015-1308-6 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kolli V, Dodds ED (2014) Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment. Analyst 139:2144–2153CrossRefPubMedGoogle Scholar
  23. 23.
    Dodds ED (2012) Gas-phase dissociation of glycosylated peptides ions. Mass Spectrom Rev 31:666–682CrossRefPubMedGoogle Scholar
  24. 24.
    Mechref Y (2012) Use of CID/ETD mass spectrometry to analyze glycopeptides. Curr Protoc Protein Sci suppl 68:Unit 12.11Google Scholar
  25. 25.
    Wuhrer M, Catalina MI, Deelder AM, Hokke CH (2007) Glycoproteomics based on tandem mass spectrometry of glycopeptides. J Chromatogr B 849:115–128CrossRefGoogle Scholar
  26. 26.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  27. 27.
    Wuhrer M, Koeleman CAM, Hokke CH et al (2005) Protein glycosylation analyzed by normal-phase of glycopeptides. Anal Chem 77:886–894CrossRefPubMedGoogle Scholar
  28. 28.
    Kolarich D, Weber A, Turecek PL et al (2006) Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms. Proteomics 6:3369–3380CrossRefPubMedGoogle Scholar
  29. 29.
    Pabst M, Altmann F (2008) Influence of electrosorption, solvent, temperature, and ion polarity on the performance of LC-ESI-MS using graphitic carbon for acidic oligosaccharides. Anal Chem 80:7534–7542CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Kathrin Stavenhagen
    • 1
    • 2
  • Hannes Hinneburg
    • 3
    • 4
  • Daniel Kolarich
    • 5
  • Manfred Wuhrer
    • 1
    • 2
    Email author
  1. 1.Division of BioAnalytical ChemistryVU University AmsterdamAmsterdamThe Netherlands
  2. 2.Center for Proteomics and MetabolomicsLeiden University Medical CenterLeidenThe Netherlands
  3. 3.Department of Biomolecular SystemsMax Planck Institute of Colloids and InterfacesPotsdamGermany
  4. 4.Department of Biology, Chemistry, Pharmacy, Institute of Chemistry and BiochemistryFreie Universität BerlinBerlinGermany
  5. 5.Department of Biomolecular SystemsMax Planck Institute of Colloids andInterfacesPotsdamGermany

Personalised recommendations