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

Research output: Contribution to journalArticle

Abstract

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant transsialidase 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 Man8-16GlcNAc2, but also mono- and di-phosphorylated oligosaccharides in the forms of Man9-15P1GlcNAc2 and Man9-14P2GlcNAc2, 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.

Original languageEnglish
Pages (from-to)1350-1361
Number of pages12
JournalGlycobiology
Volume25
Issue number12
DOIs
Publication statusPublished - Dec 1 2015

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Keywords

  • Mass spectrometry
  • N-glycan
  • P. pastoris
  • T. rangeli sialidase mutant Tr7
  • Trans-sialidase activity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Li, H., Rasmussen, M. I., Larsen, M. R., Guo, Y., Jers, C., Palmisano, G., Mikkelsen, J. D., & Kirpekar, F. (2015). Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics. Glycobiology, 25(12), 1350-1361. https://doi.org/10.1093/glycob/cwv063