title: Liquid chromatography-mass spectrometry structural characterization of neo glycoproteins aiding the rational design and synthesis of a novel glycovaccine for protection against tuberculosis.
abstract: Hereby we describe a pilot study for the rational design and synthesis of a glycoconjugate vaccine against Tuberculosis (TB) by site-specific coupling of well-defined glycans to non-antigenic amino acids in a selected protein carrier. A combination of ESI-MS and LC-MS analytical methods was applied for the systematic characterization of the reactivity of the surface amino acids in the glycosylation reaction with monosaccharides towards 2-iminomethoxyethyl or homobifunctional (4-nitrophenyl ester) linkers, both on the model protein, ribonuclease A (RNase A) and on TB10.4, the simplest antigenic protein isolated from Mycobacterium tuberculosis (MTB). Intact protein analysis was carried out to quantify the glycosylation degree and profile the glycoform composition of all the prepared neo glycoconjugates, while pronase and chymotriptic digests were analyzed to map and rank the reactivity of protein residues. Neo glycopeptides were purified by on-line porous graphitized carbon solid-phase extraction, separated by hydrophilic interaction liquid chromatography and analyzed by electrospray mass spectrometry (ESI-MS(n)). Significantly, different site specificity and glycosylation efficiency were demonstrated for the two linkers, resulting in structurally diverse glycoconjugates. A computational analysis of the amino acids involved in the epitope formation in TB10.4 addressed the choice to 2-iminomethoxyethyl-saccharide activation, that resulted in a more targeted and selective conjugation preserving the protein antigenicity. Additionally, a rational design of experiments lead to the identification of suitable experimental conditions for the preparation of highly pure and homogeneous neo glycoconjugates.
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Temporini
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy.
authors.forename: Teodora
authors.initials: T
authors.lastname: Bavaro
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Tengattini
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy.
authors.forename: Immacolata
authors.initials: I
authors.lastname: Serra
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Marrubini
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Calleri
authors.affiliation: Department of Organic and Industrial Chemistry, University of Milan, Milan, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Fasanella
authors.affiliation: Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy; The Protein Factory, Interuniversity Centre Politecnico di Milano, ICRM CNR Milano and University of Insubria, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Piubelli
authors.affiliation: Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy; The Protein Factory, Interuniversity Centre Politecnico di Milano, ICRM CNR Milano and University of Insubria, Italy.
authors.forename: Flavia
authors.initials: F
authors.lastname: Marinelli
authors.affiliation: Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy; The Protein Factory, Interuniversity Centre Politecnico di Milano, ICRM CNR Milano and University of Insubria, Italy.
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.affiliation: Department of Organic and Industrial Chemistry, University of Milan, Milan, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Speranza
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Massolini
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Italy. Electronic address: marco.terreni@unipv.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Terreni
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis; D017434:Protein Structure, Tertiary; D052616:Solid Phase Extraction / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D032581:Tuberculosis Vaccines
chemical_list: D000939:Epitopes; D006020:Glycopeptides; D006023:Glycoproteins; D032581:Tuberculosis Vaccines
keywords: Defined conjugation; Glycovaccines; Neo glycoproteins synthesis and characterization; On-line SPE-HILIC; Tuberculosis