Jean-Luc Veuthey

Identification and Functional Expression of the Mitochondrial Pyruvate Carrier

Letting Pyruvate In Transport of pyruvate is an important event in metabolism whereby the pyruvate formed in glycolysis is transported into mitochondria to feed into the tricarboxylic acid cycle (see the Perspective by Murphy and Divakaruni). Two groups have now identified proteins that are components of the mitochondrial pyruvate transporter. Bricker et al. (p. 96, published online 24 May) found that the proteins mitochondrial pyruvate carrier 1 and 2 (MPC1 and MPC2) are required for full pyruvate transport in yeast and Drosophila cells and that humans with mutations in MPC1 have metabolic defects consistent with loss of the transporter. Herzig et al. (p. 93, published online 24 May) identified the same proteins as components of the carrier in yeast. Furthermore, expression of the mouse proteins in bacteria conferred increased transport of pyruvate into bacterial cells. Two components of the mitochondrial pyruvate transporter confer transport activity when expressed in bacteria. The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.

Drug–protein binding: a critical review of analytical tools

AbstractThe extent of drug binding to plasma proteins, determined by measuring the free active fraction, has a significant effect on the pharmacokinetics and pharmacodynamics of a drug. It is therefore highly important to estimate drug-binding ability to these macromolecules in the early stages of drug discovery and in clinical practice. Traditionally, equilibrium dialysis is used, and is presented as the reference method, but it suffers from many drawbacks. In an attempt to circumvent these, a vast array of different methods has been developed. This review focuses on the most important approaches used to characterize drug–protein binding. A description of the principle of each method with its inherent strengths and weaknesses is outlined. The binding affinity ranges, information accessibility, material consumption, and throughput are compared for each method. Finally, a discussion is included to help users choose the most suitable approach from among the wealth of methods presented. FigureRange of binding constants (log Ka) assessable by the main separative and non-separative analytical tools used to characterize drug-protein interactions. ED: equilibrium dialysis, UF: ultrafiltration, PAMPA: parallel artificial membrane permeability assay, HPAC/ZE: high-performance affinity chromatography/zonal elution approach, HPAC/FA: high-performance affinity chromatography/frontal analysis approach, ACE: affinity capillary electrophoresis (mobility shift assay), CE/FA: capillary electrophoresis/frontal analysis, Spectro.: spectroscopic assays, ITC: isothermal titration calorimetry, comp.: competition studies, titration: titration studies, DSC: differential scanning calorimetry, SPR: surface plasmon resonance-based assays.

Analysis of anticancer drugs: a review.

In the last decades, the number of patients receiving chemotherapy has considerably increased. Given the toxicity of cytotoxic agents to humans (not only for patients but also for healthcare professionals), the development of reliable analytical methods to analyse these compounds became necessary. From the discovery of new substances to patient administration, all pharmaceutical fields are concerned with the analysis of cytotoxic drugs. In this review, the use of methods to analyse cytotoxic agents in various matrices, such as pharmaceutical formulations and biological and environmental samples, is discussed. Thus, an overview of reported analytical methods for the determination of the most commonly used anticancer drugs is given.

Atmospheric pressure photoionization for coupling liquid-chromatography to mass spectrometry: A review

This review presents the state-of-the-art techniques that couple liquid chromatography (LC) and mass spectrometry (MS) via atmospheric pressure photoionization (APPI). The different ionization mechanisms are discussed as well as the influence of the mobile phase composition, the nature of the dopant, etc. A comparison with other ionization sources, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), is reported, and the combination of APPI with these sources is also discussed. Several applications, covering the time period of 2005-2008, for the analysis of drugs, lipids, natural compounds, pesticides, synthetic organics, petroleum derivatives, and other substances are presented.

Intact protein analysis in the biopharmaceutical field

In recent years, a growing number of biopharmaceutical proteins have been produced and are already available, or will be soon available, in the market. These molecules are more complex to analyze than conventional low molecular weight drugs, and thus need powerful analytical approaches for the entire development and delivery process. This review summarizes the analytical techniques available for intact protein determination and the main development steps in which they are applicable. A strong emphasis has been put on separation techniques, liquid chromatography and electrophoretic techniques, but mass spectrometry and spectroscopic approaches are also mentioned. Overall, we highlight how several analytical strategies are necessary to obtain global information.

Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds.

Currently, columns packed with sub-2 μm particles are widely employed in liquid chromatography but are scarcely used in supercritical fluid chromatography. The goal of the present study was to compare the performance, possibilities and limitations of both ultra-high performance liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC) using columns packed with sub-2 μm particles. For this purpose, a kinetic evaluation was first performed, and van Deemter curves and pressure plots were constructed and compared for columns packed with hybrid silica stationary phases composed of 1.7 and 3.5 μm particles. As expected, the kinetic performance of the UHPSFC method was significantly better than that of the UHPLC. Indeed, the h(min) values were in the same range with both strategies and were between 2.2 and 2.8, but u(opt) was increased by a factor of >4 in UHPSFC conditions. Another obvious advantage of UHPSFC over UHPLC is related to the generated backpressure, which is significantly lower in the presence of a supercritical or subcritical fluid. However, the upper pressure limit of the UHPSFC system was only ∼400 bar vs. ∼1000 bar in the UHPLC system, which prevents the use of highly organic mobile phases at high flow rates in UHPSFC. Second, the impact of reducing the particle size (from 3.5 to 1.7 μm) was evaluated in both UHPLC and UHPSFC conditions. The effect of frictional heating on the selectivity was demonstrated in UHPLC and that of fluid density or decompression cooling was highlighted in UHPSFC. However, in both cases, a change in selectivity was observed for only a limited number of compounds. Third, various types of column chemistries packed with 1.7 μm particles were evaluated in both UHPLC and UHPSFC conditions using a model mixture of acidic, neutral and basic compounds. It has been shown that more drastic changes in selectivity were obtained using UHPSFC columns compared to those obtained by changing UHPLC columns. In addition, there was a good complementarity between the two separation modes. Finally, by combining the use of small particles with supercritical fluids as a mobile phase, it was possible to achieve the analysis of pharmaceutical compounds in less than 1 min or to attain a peak capacity of more than 250 in approximately 40 min, both with a high degree of repeatability.

Knowledge discovery in metabolomics: An overview of MS data handling

While metabolomics attempts to comprehensively analyse the small molecules characterising a biological system, MS has been promoted as the gold standard to study the wide chemical diversity and range of concentrations of the metabolome. On the other hand, extracting the relevant information from the overwhelming amount of data generated by modern analytical platforms has become an important issue for knowledge discovery in this research field. The appropriate treatment of such data is therefore of crucial importance in order, for the data, to provide valuable information. The aim of this review is to provide a broad overview of the methodologies developed to handle and process MS metabolomic data, compare the samples and highlight the relevant metabolites, starting from the raw data to the biomarker discovery. As data handling can be further separated into data processing, data pre-treatment and data analysis, recent advances in each of these steps are detailed separately.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.

Theory and practice of size exclusion chromatography for the analysis of protein aggregates

Size exclusion chromatography (SEC) is a historical technique widely employed for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of aggregates. The main advantage of this approach is the mild mobile phase conditions that permit the characterization of proteins with minimal impact on the conformational structure and local environment. Despite the fact that the chromatographic behavior and peak shape are hardly predictable in SEC, some generic rules can be applied for SEC method development, which are described in this review. During recent years, some improvements were introduced to conventional SEC that will also be discussed. Of these new SEC characteristics, we discuss (i) the commercialization of shorter and narrower columns packed with reduced particle sizes allowing an improvement in the resolution and throughput; (ii) the possibility of combining SEC with various detectors, including refractive index (RI), ultraviolet (UV), multi-angle laser light scattering (MALLS) and viscometer (IV), for extensive characterization of protein samples and (iii) the possibility of hyphenating SEC with mass spectrometry (MS) detectors using an adapted mobile phase containing a small proportion of organic modifiers and ion-pairing reagents.

New trends in extraction, identification and quantification of artemisinin and its derivatives

Artemisinin, a sesquiterpene lactone endoperoxide, and a number of its precursors, metabolites and semisynthetic derivatives have shown to possess antimalarial properties. Several methods have been reported for the measurement of artemisinin and its main derivatives in plant material and biological fluids. However, most of them are either not sufficiently sensitive and do not offer reliable results, or are difficult to apply in routine analyses. Therefore, new methods for the determination of these compounds, such as supercritical fluid extraction and chromatography, pressurized solvent extraction, microwave-assisted extraction, high-performance liquid chromatography coupled to mass spectrometry or evaporative light scattering detection, will be presented. Applications to plant material, pharmaceutical formulations and biological fluids will also be reviewed.