Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics.

Haiying Li, Morten I Rasmussen, Martin R Larsen, Yao Guo, Carsten Jers, Giuseppe Palmisano, Jørn D Mikkelsen, Finn Kirpekar

Index: Glycobiology 25 , 1350-61, (2015)

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Abstract

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man(8-16)GlcNA(c2), but also mono- and di-phosphorylated oligosaccharides in the forms of Man(9-15)P(1)GlcNA(c2) and Man(9-14)P(2)GlcNA(c2), respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastoris-expressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7.© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.