CE-ICP-SFMS for the detection of S and Zn in Aeromonas hydrophila Zn-beta-lactamase

Aeromonads are microorganisms that can cause both human and animal infections and is the known source of hospital-acquired infection. Some Aeromonas strains produce metallo-beta-lactamase enzymes, which are at the origin of beta-lactam resistance in members of this genus. The metallo-beta-lactamases are clinically relevant because of their ability to hydrolyse carbapenem antibiotics, and they also represent a relevant investigational model for studying molecular class B beta-lactamases because of their unique enzymological behaviour.(1,2) The Aeromonas hydrophila metallo-beta-lactamase contains Zn as enzymatic cofactor.(3-5) In this work, Zn bound to the metallo-beta-lactamase is separated from free Zn ions by capillary electrophoresis (CE) and the elements Zn and S are detected and quantified simultaneously by use of an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) operated at medium mass resolution. The goal of this investigation is to develop a method that allows a fast and accurate determination of the Zn/protein ratio. This ratio can be calculated from the Zn/S ratio. The quantification method is limited to proteins where the sulphur stoichiometry is known. The A. hydrophila Zn-beta-lactamase investigated in this work is known to contain 4 methionines and 1 cysteine. For proteins of which the primary structure is unknown, additional structural information is necessary. In this work A. hydrophila Zn-beta-lactamase is being used as proof of concept. Optimum conditions for CE, ICP-SFMS and their hyphenation were investigated. The use of the sodium salt of phytic acid as buffer additive is discussed into more detail