Challenges of determining o-glycopeptide heterogeneity: A fungal glucanase model system

Maja N. Christiansen, Daniel Kolarich, Helena Nevalainen, Nicolle H. Packer, Pia Hønnerup Jensen

Research output: Contribution to journalArticlepeer-review

40 Citations (Scopus)


O-Linked glycosylation often occurs in mucin-type domains that are heavily and heterogeneously glycosylated and are challenging to analyze. The analysis of these domains is often overlooked because of these difficulties, but changes in mucinlike domain glycosylation are implicated in many diseases. Here we have explored several strategies to determine the heterogeneity of mucinlike O-glycosylated domains. Four glucanases secreted in large quantities from Trichoderma reesei, all containing heavily O-glycosylated mucinlike linker regions, were used as a model system. The strategies involved monosaccharide compositional analysis and identification of the released glycans by HPAEC-PAD and carbon-LC ESI-MS/MS. Glycosylated peptides were generated by different protease digestions (trypsin, papain, Asp-N, PreTAQ) and enriched by HILIC microcolumns, to determine the glycopeptide heterogeneity and glycosylation sites. The complex O-glycan heterogeneity on the intact glycoproteins and the enriched mucin-type domains was determined by MALDI-MS and ESI-MS, but the dense O-glycosylation in the mucin-type domains conferred high resistance to protease cleavage. ETD-MS/MS of the glycopeptide-enriched protease digests was unsuccessful for the de novo assignment of O-glycosylation at individual sites within the mucin-type domains but allowed several previously unknown O-linked sites outside the defined linker region to be found on two of the four glucanases. The protease digests produced many glycopeptides as determined by CID-MS/MS, but ETD fragmentation of these resulted in only a few interpretable spectra, suggesting that the use of ETD for determining the heterogeneous O-glycosylation at specific sites in regions of multiple occupancy is still in its infancy.

Original languageEnglish
Pages (from-to)3500-3509
Number of pages10
JournalAnalytical Chemistry
Issue number9
Publication statusPublished - 1 May 2010


Dive into the research topics of 'Challenges of determining o-glycopeptide heterogeneity: A fungal glucanase model system'. Together they form a unique fingerprint.

Cite this