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Determination of site-specific glycan heterogeneity on glycoproteins

Nature Protocols volume 7, pages 1285–1298 (2012)Cite this article

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Abstract

The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3–5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.

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Figure 1: Overview of the techniques providing comprehensive glycoprotein characterization.
Figure 2: Schematic examples for tubing connections.
Figure 3: MS/MS spectrum of a tryptic glycopeptide derived from IgG1.
Figure 4: Selected tryptic glycopeptides from rHuEPO.
Figure 5: Glycopeptide analysis of human IgG.
Figure 6: LC-MS/MS of α1-proteinase inhibitor.

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Acknowledgements

D.K. was supported by an Erwin Schrödinger Fellowship from the Austrian Science Fund (grant J2661) and Macquarie University. P.H.J. was supported by the Danish Agency for Science, Technology and Innovation (grant 272-07-0066). The technical help of T. Dalik is as always highly appreciated.

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Author notes

  1. Daniel Kolarich & Pia H Jensen

    Present address: Present addresses: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany (D.K.); Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark (P.H.J.).,

Authors and Affiliations

  1. Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Sydney, New South Wales, Australia

    Daniel Kolarich, Pia H Jensen & Nicolle H Packer

  2. Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria

    Friedrich Altmann

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  1. Daniel Kolarich
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Contributions

D.K. performed the experiments on the therapeutic proteins and developed the initial protocol. F.A. and D.K. crucially validated MS data interpretation. D.K. and P.H.J. performed the experiments on the protein complex mixtures and optimized the protocol. D.K. and N.H.P. wrote the paper and prepared it for submission.

Corresponding author

Correspondence to Nicolle H Packer.

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The authors declare no competing financial interests.

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Kolarich, D., Jensen, P., Altmann, F. et al. Determination of site-specific glycan heterogeneity on glycoproteins. Nat Protoc 7, 1285–1298 (2012). https://doi.org/10.1038/nprot.2012.062

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