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

Pia H. Jensen, Niclas G. Karlsson, Daniel Kolarich, Nicolle H. Packer*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    222 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)1299-1310
    Number of pages12
    JournalNature Protocols
    Volume7
    Issue number7
    DOIs
    Publication statusPublished - Jul 2012

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