A ferrocene-based reagent for the conjugation and quantification of reactive metabolites.

In the present study, a method for the analysis of reactive metabolites via liquid chromatography (LC) with inductively coupled plasma-mass spectrometry (MS) was developed. A ferrocenyl-modified glutathione (GSH) reagent, consisting of GSH and succinimidyl-3-ferrocenylpropionate, was synthesized. Derivatization of the tripeptide was performed at the N-terminus, leaving the nucleophilic thiol group vacant for the attack of electrophilic compounds. The potential of ferrocenylpropionate (FP)-GSH as a trapping agent for reactive metabolites was investigated using an electrochemical flow-through cell for metabolism simulation coupled online to a LC system with electrospray ionization mass spectrometric detection. The pharmaceuticals amodiaquine, an antimalarial agent, and clozapine, an antipsychotic compound, served as model substances. By proving the successful adduct formation between the reactive metabolite and ferrocene-labeled GSH, it could be shown that FP-GSH is an effective trapping agent which eases routine reversed-phase LC analyses. In contrast to GSH, which is usually used for the conjugation of reactive metabolites and where the resulting adducts often show no or only very little retention, FP-GSH facilitates the detection of the corresponding metabolite adducts due to higher retention times.

Quantification of phytochelatins in Chlamydomonas reinhardtii using ferrocene-based derivatization.

A method for the identification and quantification of canonic and isoforms of phytochelatins (PCs) from Chlamydomonas reinhardtii was developed. After disulfide reduction with tris(2-carboxyethyl)phosphine (TCEP) PCs were derivatized with ferrocenecarboxylic acid (2-maleimidoyl)ethylamide (FMEA) in order to avoid oxidation of the free thiol functions during analysis. Liquid chromatography (LC) coupled to electrospray mass spectrometry (ESI-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) was used for rapid and quantitative analysis of the precolumn derivatized PCs. PC(2-4), CysGSH, CysPC(2-4), CysPC(2)desGly, CysPC(2)Glu and CysPC(2)Ala were determined in the algal samples depending on the exposure of the cells to cadmium ions.

Organometallic derivatizing agents in bioanalysis.

Over the last few decades, the development of several innovative hyphenated analytical techniques and their routine use in laboratories has led to new possibilities for the quantitative analysis of biomolecules. Today, the identification and quantification of biomolecules such as peptides and proteins are essential to answer important medical, pharmaceutical, and biological questions. To allow efficient detection and structure elucidation of biomolecules, several approaches including derivatization strategies were investigated and applied during recent years. This article summarizes the current approaches for labeling and presents the different types of organometallic derivatizing agents used as labels. Furthermore, their analytical potential with respect to quantification and structure elucidation for different applications in the field of bioanalysis is discussed.

Liquid chromatography with complementary electrospray and inductively coupled plasma mass spectrometric detection of ferrocene-labelled peptides and proteins.

Succinimidylferrocenyl propionate (SFP) is introduced as labelling agent for amino functions in peptides and proteins. The resulting derivatives are characterised by considerably lower polarity compared with the native analytes and can thus be well separated by means of reversed phase liquid chromatography (RP-LC). The reaction products are characterised by electrospray ionisation mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). A further advantage of the method is a simple and straightforward derivatisation protocol. Different basic and acidic model proteins as lysozyme, beta-lactoglobulin A and insulin were derivatised using SFP. Furthermore, the first dual-labelling strategy of thiol and amino groups with ferrocene-based reagents is presented. Whereas the amino groups were derivatised with SFP, the thiol groups were functionalised by reaction with ferrocenecarboxylic acid(2-maleimidoyl)ethylamide. Again, LC/ESI-MS is a suitable tool to characterise the modified peptides and proteins.

Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry.

A liquid chromatography (LC)/mass spectrometry method was developed for the determination of selected biogenic amines in various fish and other food samples. It is based on a precolumn derivatization of the amines with succinimidylferrocenyl propionate under formation of the respective amides and their reversed-phase liquid-chromatographic separation with subsequent electrospray ionization mass-spectrometric detection. Deuterated putescine, cadaverine, and histamine are added prior to the derivatization as internal standards that are coeluted, thus allowing excellent reproducibility of the analysis to be achieved. Depending on the analyte, the limits of detection were between 1.2 and 19.0 mg/kg, covering between 2 and 3 decades of linearity. The limit of detection and the linear range for histamine are suitable for the surveillance of the only defined European threshold for biogenic amines in fish samples. Compared with the established ortho-phthalaldehyde (OPA)/LC/fluorescence method, the newly developed method allows an unambiguous identification of the biogenic amines by their mass spectra in addition to only retention times, a fivefold acceleration of the separation, and independency from the sample matrix owing to the isotope-labeled internal standards. Various fish, calamari, and salami samples were successfully analyzed with the new method and validated with an independent OPA/LC/fluorescence method.

Engineering proteins with tunable thermodynamic and kinetic stabilities.

It is widely recognized that enhancement of protein stability is an important biotechnological goal. However, some applications at least, could actually benefit from stability being strongly dependent on a suitable environment variable, in such a way that enhanced stability or decreased stability could be realized as required. In therapeutic applications, for instance, a long shelf-life under storage conditions may be convenient, but a sufficiently fast degradation of the protein after it has performed the planned molecular task in vivo may avoid side effects and toxicity. Undesirable effects associated to high stability are also likely to occur in food-industry applications. Clearly, one fundamental factor involved here is the kinetic stability of the protein, which relates to the time-scale of the irreversible denaturation processes and which is determined to some significant extent by the free-energy barrier for unfolding (the barrier that "separates" the native state from the highly-susceptible-to-irreversible-alterations nonnative states). With an appropriate experimental model, we show that strong environment-dependencies of the thermodynamic and kinetic stabilities can be achieved using robust protein engineering. We use sequence-alignment analysis and simple computational electrostatics to design stabilizing and destabilizing mutations, the latter introducing interactions between like charges which are screened out at high salt. Our design procedures lead naturally to mutating regions which are mostly unstructured in the transition state for unfolding. As a result, the large salt effect on the thermodynamic stability of our consensus plus charge-reversal variant translates into dramatic changes in the time-scale associated to the unfolding barrier: from the order of years at high salt to the order of days at low salt. Certainly, large changes in salt concentration are not expected to occur in biological systems in vivo. Hence, proteins with strong salt-dependencies of the thermodynamic and kinetic stabilities are more likely to be of use in those cases in which high-stability is required only under storage conditions. A plausible scenario is that inclusion of high salt in liquid formulations will contribute to a long protein shelf-life, while the lower salt concentration under the conditions of the application will help prevent the side effects associated with high-stability which may potentially arise in some therapeutic and food-industry applications. From a more general viewpoint, this work shows that consensus engineering and electrostatic engineering can be readily combined and clarifies relevant aspects of the relation between thermodynamic stability and kinetic stability in proteins.