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Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics.
- Source :
-
Glycobiology . Dec2015, Vol. 25 Issue 12, p1350-1361. 12p. - Publication Year :
- 2015
-
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. pastorisexpressed 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 09596658
- Volume :
- 25
- Issue :
- 12
- Database :
- Academic Search Index
- Journal :
- Glycobiology
- Publication Type :
- Academic Journal
- Accession number :
- 110833200
- Full Text :
- https://doi.org/10.1093/glycob/cwv063