LC-MS Analysis of (Glyco-)Proteins of Pichia pastoris

  • Clemens Grünwald-Gruber
  • Friedrich AltmannEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)


In this chapter, we will present two methods for comprehensive glycoprotein characterization that are particularly but not exclusively useful for Pichia pastoris glycoproteins. One approach is intact protein mass measurement, where deglycosylation may be used to determine the mass of the unmodified protein. The other method is the classical bottom-up approach, where peptides and glycopeptides are analyzed by reversed-phase chromatography and detected by electrospray ionization mass spectrometry. The choice of chromatography solvents with a high ionic strength simplifies the identification of peaks of a particular peptide’s glycopattern as it leads to co-elution of neutral and charged, i.e., phosphorylated, glycoforms.

Key words

Ammonium formate buffer Mass spectrometry Electrospray ionization (ESI) Reversed-phase chromatography Monolithic protein column Phosphorylation 


  1. 1.
    Gasser B, Prielhofer R, Marx H et al (2013) Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol 8:191–208CrossRefGoogle Scholar
  2. 2.
    Wiśniewski JR, Zougman A, Nagaraj N et al (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–362CrossRefGoogle Scholar
  3. 3.
    Botelho D, Wall MJ, Vieira DB et al (2010) Top-down and bottom-up proteomics of SDS-containing solutions following mass-based separation. J Proteome Res 9:2863–2870CrossRefGoogle Scholar
  4. 4.
    Grünwald-Gruber C, Thader A, Maresch D et al (2017) Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry. Anal Bioanal Chem 409(10):2519–2530CrossRefGoogle Scholar
  5. 5.
    Huhn C, Selman MH, Ruhaak LR et al (2009) IgG glycosylation analysis. Proteomics 9:882–913CrossRefGoogle Scholar
  6. 6.
    Zauner G, Selman MH, Bondt A et al (2013) Glycoproteomic analysis of antibodies. Mol Cell Proteomics 12:856–865CrossRefGoogle Scholar
  7. 7.
    Pabst M, Altmann F (2011) Glycan analysis by modern instrumental methods. Proteomics 11:631–643CrossRefGoogle Scholar
  8. 8.
    Pabst M, Chang M, Stadlmann J et al (2012) Glycan profiles of the 27 N-glycosylation sites of the HIV envelope protein CN54gp140. Biol Chem 393:719–730CrossRefGoogle Scholar
  9. 9.
    Alagesan K, Khilji SK, Kolarich D et al (2017) It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment. Anal Bioanal Chem 2:529–538CrossRefGoogle Scholar
  10. 10.
    Hinneburg H, Hofmann J, Struwe WB et al (2016) Distinguishing N-acetylneuraminic acid linkage isomers on glycopeptides by ion mobility-mass spectrometry. Chem Commun (Camb) 52:4381–4384CrossRefGoogle Scholar
  11. 11.
    Rußmayer H, Buchetics M, Gruber C et al (2015) Systems-level organization of yeast methylotrophic lifestyle. BMC Biol 13:80CrossRefGoogle Scholar
  12. 12.
    Scott NE, Parker BL, Connolly AM et al (2011) Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni. Mol Cell Proteomics 10:M000031-MCP000201CrossRefGoogle Scholar
  13. 13.
    Irungu J, Go EP, Zhang Y et al (2008) Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein. J Am Soc Mass Spectrom 19:1209–1220CrossRefGoogle Scholar
  14. 14.
    Jez J, Castilho A, Grass J et al (2013) Expression of functionally active sialylated human erythropoietin in plants. Biotechnol J 8:371–382CrossRefGoogle Scholar
  15. 15.
    Wu SW, Pu TH, Viner R et al (2014) Novel LC-MS(2) product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides. Anal Chem 86:5478–5486CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Austrian Centre of Industrial Biotechnology (acib)ViennaAustria
  2. 2.Department of ChemistryUniversity of Natural Resources and Life Sciences Vienna (BOKU)ViennaAustria

Personalised recommendations