title: Protein haptenation by amoxicillin: high resolution mass spectrometry analysis and identification of target proteins in serum.
abstract: Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including β-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, C.S.I.C., Ramiro de Maeztu, 9, 28040 Madrid, Spain.
authors.forename: Adriana
authors.initials: A
authors.lastname: Ariza
authors.forename: Davide
authors.initials: D
authors.lastname: Garzon
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Abánades
authors.forename: Vivian
authors.initials: V
authors.lastname: de los Ríos
authors.forename: Giulio
authors.initials: G
authors.lastname: Vistoli
authors.forename: María J
authors.initials: MJ
authors.lastname: Torres
authors.forename: Marina
authors.initials: M
authors.lastname: Carini
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Aldini
authors.forename: Dolores
authors.initials: D
authors.lastname: Pérez-Sala
mesh_terms: D000328:Adult; D000658:Amoxicillin / Q000009:adverse effects / Q000737:chemistry* / Q000276:immunology / Q000493:pharmacokinetics; D000900:Anti-Bacterial Agents / Q000009:adverse effects / Q000737:chemistry* / Q000276:immunology / Q000493:pharmacokinetics; D001798:Blood Proteins / Q000737:chemistry* / Q000276:immunology; D004342:Drug Hypersensitivity / Q000276:immunology; D006241:Haptens / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008297:Male; D013058:Mass Spectrometry
chemical_list: D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D006241:Haptens; D000658:Amoxicillin