Liquid chromatography-mass spectrometry structural characterization of neo glycoproteins aiding the rational design and synthesis of a novel glycovaccine for protection against tuberculosis.

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.