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

作者：Li Haiying; Rasmussen Morten I; Larsen Martin R; Guo Yao; Jers Carsten; Palmisano Giuseppe; Mikkelsen Jorn D; Kirpekar Finn^*

来源：Glycobiology, 2015, 25(12): 1350-1361.

DOI：10.1093/glycob/cwv063

• mass spectrometry
• N-glycan
• P. pastoris
• trans-sialidase activity
• T. rangeli sialidase mutant Tr7

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 Man(8-16)GlcNAc(2), but also mono- and di-phosphorylated oligosaccharides in the forms of Man(9-15)P(1)GlcNAc(2) and Man(9-14)P(2)GlcNAc(2), 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.

• 出版日期2015-12