Differential protein labeling based on electrochemically generated reactive intermediates.

A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as ß-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identification experiments.

Electrochemistry/mass spectrometry as a tool in metabolism studies-a review.

The combination of electrochemistry (EC) and mass spectrometry (MS) has become a more and more frequently used approach in metabolism studies in the last decade. This review provides insight into the importance of metabolism studies during the drug development process and gives a short overview about the conventionally used methods since electrochemistry is often intended to substitute or minimize animal-based studies. The optimization of the electrochemical conditions is of great importance for a successful comparison with in vitro approaches. The type of metabolism reactions, which can be simulated by EC, has been extended with new cell types and working electrodes. Although the mechanism differs from the enzyme-catalyzed turnover, electrochemistry can be used to simulate a significant number of the respective reactions. An expanded set-up consisting of EC, a chromatographic separation and MS allows to distinguish between an electrospray ionization (ESI) in-source and an electrochemical oxidation and provides information on the polarity of the electrogenerated compounds. A main advantage of EC for metabolite generation is the possibility to isolate reactive species because of the purely instrumental approach. Especially when a preparative electrochemical cell with a larger working electrode surface is used, metabolites can be generated in sufficient quantities for their subsequent structure elucidation. Besides, the compounds can also be used for selective trapping experiments with different cell components such as small peptides, proteins or DNA bases. Current and possible future developments and applications of EC are presented and discussed as well.

Nonaqueous capillary electrophoresis as separation technique to support metabolism studies by means of electrochemistry and mass spectrometry.

The first combination of electrochemistry (EC), NACE, and ESI-MS to mimic the metabolic fate of drugs is described. While the combination of EC, HPLC, and ESI-MS has been used for this purpose before, NACE is able to deliver valuable additional information about possible metabolites of harmane when compared to HPLC. In this paper, NACE is used as a comprehensive separation technique in metabolism studies of harmane, a naturally occurring monoaminooxidase inhibitor, since it exhibits beneficial properties for the separation of polar compounds. Harmane is known to be metabolized via the oxidative metabolism catalyzed by cytochrome P450 enzymes, which are the most important metabolizing superfamily of enzymes in the human liver. The application of HPLC and NACE enabled the detection of 37 products in total, with 14 different mass-to-charge ratios. A total of 31 products could be detected in HPLC-MS and 26 in NACE-MS analysis. The combination of both NACE and RP-HPLC allows the identification of significantly more potential metabolites than any of the separation techniques alone.

Liquid chromatography/mass spectrometry to study oxidative degradation of environmentally relevant pharmaceuticals by electrochemistry and ozonation.

In this work, the potential of electrochemical oxidation as a tool for the rapid prediction of transformation products in water appearing after ozonation is investigated. These two approaches were compared by choosing the two environmentally relevant model compounds diclofenac and metoprolol and comparison of their transformation products after electrochemical oxidation and treatment with ozone. Within these two approaches, certain similarities were observed in the resulting chromatograms: Six transformation products of the electrochemical oxidation of metoprolol were also detected in the ozone samples. For diclofenac two transformation products matched. Additionally, five of the electrochemically generated oxidation products were reported in literature to occur after water treatment processes. The application of a boron-doped diamond working electrode for electrochemical oxidation allowed the generation of hydroxyl radicals, which was shown by spin trapping experiments with p-chlorobenzoic acid. This allowed the generation of certain transformation products previously not obtained by electrochemical oxidation. Concluding, the hyphenation of electrochemistry with liquid chromatography and mass spectrometry offers a useful tool in transformation studies.

Influence of cimetidine and its metabolites on Cisplatin--investigation of adduct formation by means of electrochemistry/liquid chromatography/electrospray mass spectrometry.

Cimetidine has been studied as an additive in cancer chemotherapy. It is claimed to reduce the side effects of Cisplatin. This study focuses on possible interactions between Cisplatin and cimetidine on the molecular level. Due to the fact that cimetidine is metabolized in the liver, interactions between its metabolites and Cisplatin are also investigated. By means of LC/ESI-MS, Cisplatin-cimetidine adducts were detected. In a second step, the metabolism of cimetidine was simulated by electrochemical oxidation. These results were compared with microsomal incubations of cimetidine using rat and human liver cell microsomes. Because the two methods showed a correlation, the electrochemical approach was further used to investigate Cisplatin's interactions with metabolites of cimetidine. However, notable interactions that might take place in the human body could neither be observed for pure cimetidine nor for its metabolites. Finally, the impact of cimetidine on Cisplatin-protein interactions were studied using the model protein ß-lactoglobulin A. In the presence of cimetidine, the affinity of Cisplatin towards the model protein appears to be increased.

Electrochemistry/liquid chromatography/mass spectrometry to demonstrate irreversible binding of the skin allergen p-phenylenediamine to proteins.

RATIONALE: para-Phenylenediamine (PPD) is a potent and well-known allergen, which is commonly used in hair or fur dyes and can cause severe allergic contact dermatitis. In this work, the skin-sensitizing potential of PPD with respect to the conjugation of proteins was evaluated using an approach without animal testing. METHODS: Electrochemistry (EC) coupled offline to liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS) was employed to convert the pre-hapten PPD into its reactive hapten analogs. A previous study had already shown that this purely instrumental method is suitable for accelerating and simulating the various oxidation processes, which PPD may undergo, and that the emerging products are prone to react with soft thiol groups of small nucleophiles like glutathione and cysteine. RESULTS: This investigation was extended by successfully demonstrating adduct formation between EC-generated PPD oxidation products and the three proteins ß-lactoglobulin A (ß-LGA), human serum albumin and human hemoglobin. A tryptic digest of modified ß-LGA provided evidence for irreversible protein binding of monomeric PPD, a PPD dimer and the PPD trimer known as Bandrowski's Base. It was shown that the main oxidation product p-phenylene quinone diimine, and the reactive oligomerized species, primarily attack the free thiol function of proteins rather than other nucleophilic amino acid residues. CONCLUSIONS: The pre-hapten PPD was efficiently activated upon EC oxidation and the resulting species were further reacted with different proteins leading to diverse hapten-protein complexes. Thereby, problems related to the complex matrix present in conventional in vitro or in vivo methods could effectively be avoided.

Electrochemistry/mass spectrometry as a tool in the investigation of the potent skin sensitizer p-phenylenediamine and its reactivity toward nucleophiles.

RATIONALE: Although para-phenylenediamine (PPD) is known to cause severe allergic contact dermatitis in consequence of autoxidation and/or skin metabolism pathways, it is commonly utilized as an ingredient in permanent hair dyes. The aim of this work was to simultaneously accelerate the autoxidation process and to simulate the metabolic activation of PPD using a purely instrumental system. METHODS: Electrochemistry (EC) in combination with electrospray ionization mass spectrometry (ESI-MS) was used in this study to assess the skin-sensitizing potential of PPD. Online and offline coupled EC/ESI-MS experiments were carried out and the emerging oxidation products were investigated. In a second approach, these primary species were allowed to react with the nucleophiles glutathione (GSH), cysteine (Cys), potassium cyanide (KCN) and lysine (Lys) in order to evaluate their reactivity. RESULTS: The reactive p-phenylene quinone diimine (PPQD), which can form upon autoxidation and/or skin metabolism of PPD, was effectively generated in a simple EC cell next to further oxidation products, including the trimeric product Bandrowski's Base (BB). Conjugation with GSH and Cys was successfully proven, but no adducts with KCN or Lys were observed. Furthermore, the application of different concentration ratios between PPD and nucleophile was shown to play a crucial role concerning the type of oxidation products and adducts being formed. CONCLUSIONS: It was found that EC/MS is a well-suited approach for the targeted generation of reactive haptens such as PPQD while avoiding detection problems due to the complexity of matrices encountered when conducting conventional in vitro or in vivo experiments.

Identification and quantification of potential metabolites of Gd-based contrast agents by electrochemistry/separations/mass spectrometry.

Oxidative and potentially metabolic pathways of the five most frequently used contrast agents for magnetic resonance imaging (MRI) based on gadolinium (Gd) are examined. The oxidation of gadopentetate (Gd-DTPA) was studied with a focus on electrochemical oxidation coupled to analytical separation methods and mass spectrometric detection. Mass voltammograms generated with online electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) gave a first overview of oxidation products. Two potential metabolites could be detected, with the major metabolite originating from an N-dealkylation (M1). Four other Gd complexes used as MRI contrast agents showed similar reactions in the EC/ESI-MS set-up. To obtain more information about the properties and the quantity of the generated products, a wide range of separation and detection techniques was applied in further experiments. Gd-DTPA and its N-dealkylation product were successfully separated by capillary electrophoresis (CE) and detected by ESI-MS and inductively coupled plasma (ICP)-MS, respectively. CE experiments indicated that the second oxidation product (M2) detected in the mass voltammogram is unstable and decomposes to M1. Employing EC/CE/ICP-MS, the quantification of the metabolites could be achieved. Under the employed conditions, 8.8% of Gd-DTPA was oxidized. Online experiments with high performance liquid chromatography (HPLC) coupled to ESI-MS confirmed the decomposition of M2. Time-resolved measurements showed a decrease of M2 and a simultaneous increase in M1 within only a few minutes, confirming the conclusion that M2 degrades to M1, while EC/LC/ICP-MS measurements provided quantitative evidence as well. The EC/MS simulation shows that a metabolic transformation should not be disregarded in further research regarding the trigger of nephrogenic systemic fibrosis (NSF), a disease exclusively observed for several hundred dialysis patients after delivery of Gd-based MRI contrast agents with linear structure. Furthermore, the used methods may allow the prediction of options for the oxidative removal of these contrast agents from wastewaters.

Simulation of the oxidative metabolism of diclofenac by electrochemistry/(liquid chromatography/)mass spectrometry.

Diclofenac is a frequently prescribed drug for rheumatic diseases and muscle pain. In rare cases, it may be associated with a severe hepatotoxicity. In literature, it is discussed whether this toxicity is related to the oxidative phase I metabolism, resulting in electrophilic quinone imines, which can subsequently react with nucleophiles present in the liver in form of glutathione or proteins. In this work, electrochemistry coupled to mass spectrometry is used as a tool for the simulation of the oxidative pathway of diclofenac. Using this purely instrumental approach, diclofenac was oxidized in a thin layer cell equipped with a boron doped diamond working electrode. Sum formulae of generated oxidation products were calculated based on accurate mass measurements with deviations below 2 ppm. Quinone imines from diclofenac were detected using this approach. It could be shown for the first time that these quinone imines do not react with glutathione exclusively but also with larger molecules such as the model protein ß-lactoglobulin A. A tryptic digest of the generated drug-protein adduct confirms that the protein is modified at the only free thiol-containing peptide. This simple and purely instrumental set-up offers the possibility of generating reactive metabolites of diclofenac and to assess their reactivity rapidly and easily.

Electrochemistry/liquid chromatography/mass spectrometry as a tool in metabolism studies.

Electrochemistry/liquid chromatography/mass spectrometry is a powerful complementary tool for the simulation of the oxidative metabolism of drugs and other xenobiotics.

Fast method for monitoring phospholipase A2 activity by liquid chromatography-electrospray ionization mass spectrometry.

A new liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the fast determination of phospholipase A(2) (PLA(2)) activity has been developed. For the first time, the method allows the parallel detection of glycerophosphatidylcholine (GroPCho) as PLA(2) substrate as well as of its products fatty acid (FA) and lyso-GroPCho. ESI-MS was carried out in negative ion mode, detecting the FA as [M-H](-) ions and the lyso-GroPCho and GroPCho as acetate adducts [M+Ac](-). Utilizing a fast gradient on a short C(5)-modified silica gel column with 3 microm particles, five GroPChos, five FAs and six lyso-GroPChos could be separated according to their chain length in less than 3 min. A very high average chromatographic efficiency of 41,200 theoretical plates (plate height 0.5 microm) was achieved for the separation of the GroPChos. The method was applied for monitoring the release of arachidonic acid (20:4 FA) and 1-stearoyl-lyso-sn-GroPCho (18:0 GroPCho) from unilamellar vesicles of 1-stearoyl-2-arachidonoyl-sn-GroPCho (18:0/20:4 GroPCho). With a limit of detection of 0.5 pmol (total amount injected on column) for the FAs and lyso-GroPChos and 1.5 pmol for the GroPChos as well as a linear range of 1.5 decades, the method has proven to be suitable for the monitoring of different secretory PLA(2) (sPLA(2)) conversions. Furthermore, it was applied to screen a small library of PLA(2) inhibitors for their activity towards sPLA(2) type V and snake venom of Bothrops moojeni. In both cases, active samples could be directly identified. With its short analysis time, its high chromatographic efficiency and the parallel detection of substrate and all products, the developed LC-ESI-MS method is well suited for the analysis of PLA(2) activity.

Analysis of glutathione adducts of patulin by means of liquid chromatography (HPLC) with biochemical detection (BCD) and electrospray ionization tandem mass spectrometry (ESI-MS/MS).

A novel method for the identification of glutathione/electrophile adducts that are inhibiting glutathione-S-transferase (GST) activity was developed and applied for the analysis of the mycotoxin patulin. The method is based on high-performance liquid chromatography (HPLC) coupled to a continuous-flow enzyme reactor serving as biochemical detector (BCD) in parallel to electrospray mass spectrometric detection (ESI-MS). This HPLC-BCD technique combines a separation step and the detection of the inhibition and is therefore ideally suited for the analysis of the activity of single patulin/glutathione adducts within a complex mixture of adducts. Two out of at least 15 detected patulin-glutathione adducts showed strong GST inhibition. In ESI-MS, the inhibitory active adducts were characterized by [M + H]+ ions with m/z 462.1138 and m/z 741.2011, respectively. They could be identified as a dihydropyranone adduct containing one molecule glutathione and a ketohexanoic acid bearing two glutathione molecules.