Targeted profiling of polar intracellular metabolites using ion-pair-high performance liquid chromatography and -ultra high performance liquid chromatography coupled to tandem mass spectrometry: Applications to serum, urine and tissue extracts
Michopoulos F.,Oncology iMED |
Michopoulos F.,Aristotle University of Thessaloniki |
Whalley N.,Oncology iMED |
Theodoridis G.,Aristotle University of Thessaloniki |
And 3 more authors.
Journal of Chromatography A | Year: 2014
The effective analysis of polar ionic metabolites by LC-MS, such as those encountered in central carbon metabolism, represents a major problem for metabolic profiling that is not adequately addressed using strategies based on either reversed-phase or HILIC methods. Here we have compared analysis of central carbon metabolites on optimized methods using HILIC, porous graphitic carbon or ion pair chromatography (IPC) using tributyl ammonium as IP reagent. Of the 3 chromatographic approaches examined only IPC enabled us to obtain a robust analytical methodology. This system was used to profile more than a hundred endogenous metabolic intermediates in urine, serum and tissue samples. However, whilst we found IPC to be the best of the approaches examined considerable care was still needed to obtain robust data. Thus, in excess of 40 of representative biological samples were needed to "condition" a new analytical column and further 10 matrix injections were then required at the beginning of each analytical batch in order to obtain robust and reproducible chromatographic separations. An additional limitation that we have found was that, for a small number of phosphorylated and poly carboxylic acid metabolites, measurement was only possible if the analytes were present in relatively high concentrations. We also found that, whilst this methodology could be used for the analysis of both in vitro cell culture media, cell extracts, tissue, and biological fluids (blood, urine), for the best results columns should only be used to analyze a single matrix. However, despite the need for extensive column conditioning, and the manifold disadvantages resulting from the contamination of the separation system and mass spectrometer with the ion pair reagent, IPC-MS currently provides the best means of analyzing these polar, ionic and problematic metabolites. © 2014 Elsevier B.V.
Ward R.A.,Oncology IMED |
Kettle J.G.,Oncology IMED
Journal of Medicinal Chemistry | Year: 2011
The availability of suitable chemical building blocks, or reagents, is a key factor that determines the degree of effort required to make a target molecule. If a reagent is not available and requires synthesizing, this increases the total number of synthetic steps in the route and may result in a less attractive synthetic target. This can impact most in compound collection enhancement activities or early lead identification (LI) where typically not enough information or data are available to commit to such long multistep syntheses. In lead optimization (LO) projects, having access to commonly used reagents may improve the efficiency of building structure-activity relationships (SARs) and structure-property relationships (SPRs) around a core scaffold. This paper describes the systematic enumeration of key heteroaromatic reagent classes and the subsequent analysis of the availability of these in a number of commonly used databases. © 2011 American Chemical Society.
PubMed | University of Dundee, Oncology iMED and Astrazeneca
Type: | Journal: Scientific reports | Year: 2016
Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class.