Citation: CHU Jian, JIA Tian-Yuan, GONG Zhong-YING, DONG Chen-Ying, ZHOU Da-Wei. Characterization of Biosynthetic Pathway of Tetrasaccharide Repeating Unit of Escherichia Coli O77 O-antigen by Matrix-assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, ;2020, 48(8): 1096-1103. doi: 10.19756/j.issn.0253-3820.191770 shu

Characterization of Biosynthetic Pathway of Tetrasaccharide Repeating Unit of Escherichia Coli O77 O-antigen by Matrix-assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry

1.
School of Automation and Electrical Engineering, Tianjin University of Technology and Education, Tianjin 300222, China;
2.
TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin 300457, China;
3.
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin 300457, China;
4.
Department of Neurology, Tianjin First Center Hospital, Nankai University, Tianjin 300192, China

Received Date: 29 December 2019
Revised Date: 12 June 2020

Fund Project: This work was supported by the Fundamental Research Funds for the Central Universities, Nankai University (No. 63191133) and the Tianjin Enterprise Science and Technology Commissioner Project (No. 18JCTPJC65600).

Mass spectrometry is a rapid, sensitive and accurate approach for the direct monitoring of enzyme-catalyzed reaction that does not require chromophore or radiolabeling, and thus provides a viable alternative to existing analytical techniques. Herein, a simple and efficient assay for characterization of E. coli O77 O-unit biosynthetic pathway by using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) was demonstrated by WbaD and WbaC (two glycosyltransferases from E. coli O77 O antigen) lipooligosacharide enzymatic products, respectively. The rapid and direct monitoring of the enzymatic reaction was achieved by subjecting a small amount (0.3 μL) of the reaction mixture to MS analysis without chromatographic separation or desalting steps, and subsequent MS-MS analyses of their lipooligosacharide enzymatic products via collision-induced dissociation enabled the structure of the products of enzyme-catalyzed reaction to be determined. The results demonstrated that WbaD catalyzed the transfer of the first Man residue to GlcNAc-PP-PhU and formed Man-GlcNAc-PP-PhU, and WbaC acted actted after WbaD, adding two Man residues to the growing polymer. Furthermore, the model for E. coli O77 O-unit biosynthesis was studied with the help of time-lapse analysis, and it was proposed that WbaC and WbaD acted in both cooperative and alternating fashion to form the repeat unit and WbaC did not act in tandem with WbaD. Collectively, these data demonstrated that a high-energy CID MALDI-TOF/TOF MS-based platform was applicable to the facile characterization of biosynthetic pathways of O-unit and offered significant advantages over current methods in terms of speed, sensitivity, reproducibility, automation and reagent costs, which would open new way for the future mechanistic study of O-unit biosynthesis and exploitation of these fascinating glycocatalysts.
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