Expanding role of Electrochemistry/Mass spectrometry in life sciences

AGNIESZKA KRAJ
Antec, Zoeterwoude, The Netherlands


Abstract:

Electrochemistry (EC) is a complementary approach to traditional methods such as in vivo or in vitro metabolism studies, and delivers the oxidative metabolic fingerprint of a (drug) molecule in a very short time. In this lecture a dedicated electrochemical system will be presented for fast screening of drugs to obtain their metabolic fingerprint. Furthermore, a novel micro preparative electrochemical cell will be presented for highly efficient metabolite generation. The cell can be hyphenated to MS or LC/MS to perform separation and identification of the created oxidative compounds i.e., intermediates, (reactive) metabolites, etc. Alternatively the cell can be used off-line and the generated metabolites can then be collected for supplementary research such as NMR. The on-line EC/MS system allows for automated acquisition of a so called MS voltammogram. A MS voltammogram visualizes the ion abundance versus m/z as a function of applied potential to the electrochemical cell. With a MS voltammogram the optimal potential can be determined for electrochemical generation of the desired metabolites. The easy and fast electrochemical conversion of Amodiaquine (model compound) into its major phase I metabolites will be presented in both analytical and preparative cells. In a second step Glutathione (GSH) is added to the electrochemically generated metabolites to form the appropriate GSH-metabolite adducts, mimicking phase II reactions. All known adducts were successfully formed and identified with MS.

Recently, the scope of Electrochemistry (EC) upfront MS has been extended from mimicking drug metabolism towards new applications such as: protein/peptide cleavage, disulfide bonds reduction in proteins/peptides, covalent DNA adduct formation, covalent drug-protein binding, etc. In this lecture we will show the application of on-line EC/MS as a powerful tool to simulate various oxidation and reduction processes in life sciences. Electrochemistry up front MS can be applied for protein and peptide cleavage (as a promising new approach to enzymatic digestion).
Electrochemical cleavage of proteins and peptides occurs very specifically at C-terminal of the Tyrosine and Tryptophan peptide bonds. Examples of oxidative cleavage will be presented. Disulfide bonds are one of the most important posttranslational modifications for proteins. In this paper we present the structural analysis of biologically active peptides and proteins containing disulfide bonds (e.g., insulin, etc) using electrochemistry (EC) combined with mass spectrometry.
Therefore the sample undergoes electrolytic disulfide cleavage in the electrochemical flow cell followed by online MS analysis. Based on the intact protein mass and the resulting fragments and the MS/MS data unambiguous assignment of the disulfide bonds becomes possible. Investigation of drug-protein adducts by conventional techniques (microsomal incubation, in-vivo studies) are very laborious and time-consuming. With the application of EC, it is possible to activate proteins and drugs within seconds to undergo covalent drug-protein binding. EC was used to initiate adduct formation with DNA. The obtained reaction products were separated by LC and detected by MS. Tandem MS experiments were used for structural confirmation. In a proof of principle study acetaminophen was selected as model compound. Covalent adduct formation was observed for electrochemical activated mixtures of acetaminophen and guanosine.

All these applications illustrate the tremendous power and broad applicability of electrochemistry as a tool to mimic nature's Redox reactions within a few seconds that can take weeks or months in real life.

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