Structural analysis of N- and O-glycans released from glycoproteins

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Abstract

This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

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Figure 1: Screening of N-linked glycans from erythropoietin.
Figure 2: Screening of reduced N-linked and O-linked glycans from erythropoietin.
Figure 3: PGC LC-MS N-glycan profile derived from 5 μg of human IgG.
Figure 4: Separation by LC enables individual tandem-MS spectra acquisition for individual glycan structures.
Figure 5

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Acknowledgements

P.H.J. was supported by the Danish Agency for Science, Technology and Innovation (grant 272-07-0066). D.K. was supported by an Erwin Schrödinger Fellowship from the Austrian Science Fund (grant J2661) and Macquarie University. We also thank M. Nakano for the preparation of Figure 5 (based on data from ref. 5).

Author information

All authors contributed equally to this work. N.G.K. developed and validated the initial protocol and performed the analysis of recombinant EPO. P.H.J. tested, optimized and wrote the protocol. D.K. and N.H.P. co-wrote and edited the final manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.

Correspondence to Nicolle H Packer.

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Jensen, P., Karlsson, N., Kolarich, D. et al. Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc 7, 1299–1310 (2012) doi:10.1038/nprot.2012.063

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