Mass spectrometric strategies for the identification and characterization of human serum albumin covalently adducted by amoxicillin: ex vivo studies.

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Garzon D, Ariza A, Regazzoni L, Clerici R, Altomare A, Sirtori FR, Carini M, Torres MJ, Perez-Sala D, Aldini G

Mass spectrometric strategies for the identification and characterization of human serum albumin covalently adducted by amoxicillin: ex vivo studies.

Chem Res Toxicol. 2014 Sep 15;27(9):1566-74. doi: 10.1021/tx500210e. Epub 2014 Aug 13.

PubMed ID

25088930 [ View in PubMed

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Abstract

This study addresses the detection and characterization of the modification of human serum albumin (HSA) by amoxicillin (AX) in ex vivo samples from healthy subjects under oral amoxicillin administration (acute intake of 1 g every 8 h for 48 h). To reach this goal, we used an analytical strategy based on targeted and untargeted mass spectrometric approaches. Plasma samples withdrawn before AX oral intake represented the negative control samples to test the method selectivity, whereas HSA incubated in vitro with AX was the positive control. Different MS strategies were developed, particularly (1) multiple reaction monitoring (MRM) and precursor ion scan (PIS) using a HPLC system coupled to a triple quadrupole MS analyzer and (2) a dedicated data-dependent scan and a customized targeted MS/MS analysis carried out using a nano-LC system coupled to a high-resolution MS system (LTQ Orbitrap XL). Lys 190 was identified as the only modification site of HSA in the ex vivo samples. The AX adduct was identified and fully characterized by complementary targeted approaches based on triple quadrupole (MRM mode) and orbitrap (SIC mode) mass analyzers. The SIC mode also permitted the relative amount of AX-adducted HSA to be measured, ranging from 1 to 2% (6-12 muM) at 24 and 48 h after the oral intake. No adduct in any ex vivo sample was identified by the untargeted methods (PIS and data-dependent scan mode analysis). The results on one hand indicate that MS, in particular high-resolution MS, analysis represents a suitable analytical tool for the identification/characterization of covalently modified proteins/peptides; on the other hand, they give deeper insight into AX-induced protein haptenation, which is required to better understand the mechanisms involved in AX-elicited allergic reactions.

DrugBank Data that Cites this Article

Drug Carriers

Drug	Carrier	Kind	Organism	Pharmacological Action	Actions
Amoxicillin	Serum albumin	Protein	Humans	Unknown	Binder	Details