title: A novel mass spectrometric strategy "BEMAP" reveals Extensive O-linked protein glycosylation in Enterotoxigenic Escherichia coli.
abstract: The attachment of sugars to proteins via side-chain oxygen atoms (O-linked glycosylation) is seen in all three domains of life. However, a lack of widely-applicable analytical tools has restricted the study of this process, particularly in bacteria. In E. coli, only four O-linked glycoproteins have previously been characterized. Here we present a glycoproteomics technique, termed BEMAP, which is based on the beta-elimination of O-linked glycans followed by Michael-addition of a phosphonic acid derivative, and subsequent titanium dioxide enrichment. This strategy allows site-specific mass-spectrometric identification of proteins with O-linked glycan modifications in a complex biological sample. Using BEMAP we identified cell surface-associated and membrane vesicle glycoproteins from Enterotoxigenic E. coli (ETEC) and non-pathogenic E. coli K-12. We identified 618 glycosylated Serine and Threonine residues mapping to 140 proteins in ETEC, including several known virulence factors, and 34 in E. coli K-12. The two strains had 32 glycoproteins in common. Remarkably, the majority of the ETEC glycoproteins were conserved in both strains but nevertheless were only glycosylated in the pathogen. Therefore, bacterial O-linked glycosylation is much more extensive than previously thought, and is especially important to the pathogen.
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.|The ithree institute, University of Technology Sydney, PO Box 123, Broadway NSW 2007, Australia.
authors: Anders
authors: A
authors: Boysen
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.|GlycoProteomics laboratory, Department of Parasitology, University of Sao Paulo, Brazil.
authors: Giuseppe
authors: G
authors: Palmisano
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors: Thøger Jensen
authors: TJ
authors: Krogh
authors: The ithree institute, University of Technology Sydney, PO Box 123, Broadway NSW 2007, Australia.
authors: Iain G
authors: IG
authors: Duggin
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors: Martin R
authors: MR
authors: Larsen
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors: Jakob
authors: J
authors: Møller-Jensen
mesh_terms: D054307:Enterotoxigenic Escherichia coli / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D006023:Glycoproteins