RESUMEN
The direct separation of isomeric glucuronide metabolites from propranolol dosed tissue extracts by differential mobility spectrometry-mass spectrometry (DMS-MS) with the use of the polar gas-phase chemical modifier acetonitrile was demonstrated. The DMS gas-phase separation was able to resolve the isomeric metabolites with separation times on the order of milliseconds instead of minutes which is typically required when using pre-ionization chromatographic separation methods. Direct separation of isomeric metabolites from the complex tissue extract was confirmed by implementing a high-performance liquid chromatography (HPLC) separation prior to the DMS-MS analysis to pre-separate the species of interest. The ability to separate isomeric exogenous metabolites directly from a complex tissue extract is expected to facilitate the drug development process by increasing analytical throughput without the requirement for pre-ionization cleanup or separation strategies.
Asunto(s)
Descubrimiento de Drogas/métodos , Histocitoquímica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Líquida de Alta Presión , Glucurónidos/análisis , Glucurónidos/química , Glucurónidos/metabolismo , Isomerismo , Hígado/química , Hígado/metabolismo , Masculino , Ratones , Propranolol/farmacocinéticaRESUMEN
Current methods for high-throughput screening (HTS) use a serial process to evaluate compounds as inhibitors toward a single therapeutic target, but as the demand to reduce screening time and cost continues to grow, one solution is the development of multiplex technology. In this communication, the multiplex assay capability of a mass spectrometry (MS)-based readout system is verified using a kinase and esterase reaction simultaneously. Furthermore, the MS-based readout is shown to be compatible with a typical HTS workflow by identifying and validating several new inhibitors for each enzyme from a small library of compounds. These data confirm that it is possible to monitor inhibition of multiple therapeutic targets with one pass through the compound repository, thus demonstrating the potential for MS-based methods to become a method of choice for HTS of isolated enzymes.
Asunto(s)
Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de Masas/métodos , Acetilcolinesterasa/metabolismo , Calibración , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Pruebas de Enzimas/normas , Inhibidores Enzimáticos/farmacología , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas/normas , Modelos Biológicos , Bibliotecas de Moléculas Pequeñas/análisisRESUMEN
Mass spectrometry (MS)-based high-throughput screening (HTS) has tremendous potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label-free readout. We recently reported that a new generation matrix-assisted laser desorption/ionization triple quadrupole (MALDI-QqQ) mass spectrometer is ideally suited for a variety of enzyme assays and screening protocols. However, all the targets measured to date had peptide substrates that were easily monitored by selected ion monitoring (SIM) without interference from the MALDI matrix. To further extend the application to enzymes with small molecule, non-peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcholinesterase (AChE). Due to the potential of MALDI matrix interference, multiple reaction monitoring (MRM) was investigated for selective MS/MS transitions and to accurately measure the conversion of acetylcholine into choline. Importantly, ionization, detection and MRM transition efficiency differences between the substrate and product can be overcome by pre-balancing the MRM transitions during method development, thus allowing for a direct readout of the enzyme activity using the ratio of the substrate and product signals. Further validation of the assay showed accurate concentration-dependent inhibition measurements of AChE with several known inhibitors. Finally, a small library of 1008 drug-like compounds was screened at a single dose (10 microM) and the top 10 inhibitors from this primary screen were validated in a secondary screen to determine the rank order of inhibitory potency for each compound. Collectively, these data demonstrate that a MALDI-QqQMS-based readout platform is amenable to measuring small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs.
Asunto(s)
Acetilcolinesterasa/química , Inhibidores de la Colinesterasa/química , Inhibidores Enzimáticos/química , Proteínas de Peces/química , Bibliotecas de Moléculas Pequeñas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Electrophorus/metabolismo , Proteínas de Peces/antagonistas & inhibidores , Unión Proteica , Especificidad por SustratoRESUMEN
A MALDI ion source on a triple quadrupole mass spectrometer constructed for the purpose of obtaining high speed quantitative measurements on drugs and other low molecular weight compounds is described. Particular attention is given to the ion generation and transport phenomena that affect analysis speed, throughput, and practical instrument robustness. In this regard parameters that affect desorption speed, beam spreading, ion flight times, sensitivity, signal-to-noise, ion fragmentation, sample carry-over, and instrument contamination are examined and experimental results are provided. MALDI and electrospray sensitivity is compared, to provide a practical frame of reference.