Trichoderma reesei has been of industrial interest for the production of cellulases, resulting in the development of hypercellulolytic strains such as RUT-C30. High protein production and secretion, and eukaryotic glycosylation machinery, make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The N-glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients but O-glycosylation has been less extensively studied. Under glucose as a sole carbon source, secreted protein glycosylation featured novel O-glycan compositions which made up over a third of the total abundance of O-glycans released from these proteins.

This study characterised these novel compositions, confirming the presence of hexuronic acid in released O-glycans. Negative mode ESI-PGC-LC-MS/MS using a ThermoFisher Velos Pro was used to identify and characterise these O-glycans through annotation of glycosidic bond and cross-ring fragments. Trap-HCD allowed targeted MS3 experiments to be performed on the hexuronic acid substituent of these structures which was not possible with CID, validating the novel O-glycan composition. One site of novel O-glycosylation was identified, revealing attachment to a peptide in the catalytic domain of CBHI. These are the first reported glycans to contain acidic sugars in fungi and could have significant implications for CBHI structure and activity.