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 journalJournal articleResearchpeer-review

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 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
JournalGlycobiology
Volume25
Issue number12
Pages (from-to)1350-1361
ISSN0959-6658
DOIs
Publication statusPublished - Dec 2015

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Trypanosoma rangeli
Pichia
Neuraminidase
Bioinformatics
Tandem Mass Spectrometry
Computational Biology
Mass spectrometry
Polysaccharides
Oligosaccharides
Functional analysis
Glycopeptides
Liquid chromatography
Human Milk
Mannose
Liquid Chromatography
Pipelines
Chemical analysis
Proteins

Cite this

Li, Haiying ; Rasmussen, Morten I ; Larsen, Martin R ; Guo, Yao ; Jers, Carsten ; Palmisano, Giuseppe ; Mikkelsen, Jørn D ; Kirpekar, Finn. / Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics. In: Glycobiology. 2015 ; Vol. 25, No. 12. pp. 1350-1361.
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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 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.",
author = "Haiying Li and Rasmussen, {Morten I} and Larsen, {Martin R} and Yao Guo and Carsten Jers and Giuseppe Palmisano and Mikkelsen, {J{\o}rn D} and Finn Kirpekar",
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Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics. / Li, Haiying; Rasmussen, Morten I; Larsen, Martin R; Guo, Yao; Jers, Carsten; Palmisano, Giuseppe; Mikkelsen, Jørn D; Kirpekar, Finn.

In: Glycobiology, Vol. 25, No. 12, 12.2015, p. 1350-1361.

Research output: Contribution to journalJournal articleResearchpeer-review

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T1 - Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics

AU - Li, Haiying

AU - Rasmussen, Morten I

AU - Larsen, Martin R

AU - Guo, Yao

AU - Jers, Carsten

AU - Palmisano, Giuseppe

AU - Mikkelsen, Jørn D

AU - Kirpekar, Finn

N1 - © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PY - 2015/12

Y1 - 2015/12

N2 - 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 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.

AB - 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 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.

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JO - Glycobiology

JF - Glycobiology

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