Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase com position in complex fermentation samples of secreted proteins from Trichode rma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Ce17 A), a major component in these extracts, was purified from different strain s and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PA D), and capillary liquid chromatography-electrospray mass spectrometry (cLC -ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. S elective identification of tryptic N-linked glycopeptides was achieved usin g LC-ESMS on a quadrupole/time-of-flight instrument with a mixed scan funct ion. The suspected glycopeptides were further analyzed by on-line tandem ma ss spectrometry to determine the nature of N-linked glycans and their attac hment sites, This strategy enabled the identification of a high mannose gly can attached to Asn270 (predominantly Man(8)GlcNAc(2)) and single GlcNAc oc cupancy at Asn45 and Asn384 with some site heterogeneity depending on strai ns and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser resid ues as indicated by MALDI-TOF and HPAEC-PAD experiments. Additional heterog eneity was noted in the CBH I linker peptide of RUT-C30 strain with the pre sence of a phosphorylated di-saccharide. (C) 2001 Elsevier Science B.V. All rights reserved.