Liquid Chromatography-Mass Spectrometric Multiple Reaction Monitoring-based Strategies for Expanding Targeted Profiling towards Quantitative Metabolomics

Abstract

Recent advances in analytical methodologies have made it possible to bring metabolomic profiling into quantitative metabolomics that permits precise measurements of comprehensive small-molecule profiles. Modern liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) mode serves as the foundation for accurate simultaneous multi-analyte quantitation across large sample sets to provide high-quality information on target molecular profiles in complex systems. Despite the intrinsic multiplexing potential of the LC-MRM-MS technique, the key bottleneck in current LC-MRM-based assays is generally the limited analyte coverage and throughput capacity. Nowadays, the MRM-based approach has emerged as an attractive strategy for quantitative proteomic analysis and high-throughput biomarker discovery. So far, the full potential of the contemporary LCMRM methodology unleashed for quantitative metabolite profiling and metabolomic measurements of non-peptidic small molecules is rarely discussed. In this review we attempt to provide an overview on the major recent developments in LC-MRM-based strategies for quantitative profiling of multi- and non-target small molecules in biological samples. This article highlights the utility and power of the LC-MRM-based targeted approaches as valuable bioanalytical tools for low-cost, multiplexed quantitation on a large scale, with special emphasis on the promise of combining various strategies for expanding coverage and throughput of the LC-MRM-based assays to cover the gap between a widely targeted profiling and large-scale unknown screening towards comparative or quantitative metabolomics. General issues raised in metabolite profiling, such as basic aspects of bioanalysis, methodological dilemmas and challenges in quantitative metabolomics are addressed, and different strategies to circumvent the existing bottleneck and potential pitfalls of the current LC-MRM-MS techniques are outlined. In addition, the rudiments of LC-MRM-MS and its recent applications in combination with such strategies for biomarker quantitation and verification is also described.

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

“…The MRM method 25 optimization for analysis of targeted 13 Cmetabolic flux profiling of glucose and its intermediate metabolites was performed using standard aqueous stock solutions of selected metabolites, namely G6P, F6P, citrate, a-KG, fumarate, malic acid, glutamic acid and glutamine ( Fig. 1 and Table 1).…”

“…Such biomarker(s) will help in early detection and classification of neurocognitive impairment. 24 Furthermore, multiple reaction monitoring (MRM) 25 can also be applied to drug discovery assays in the context of HIV-1 infection of macrophages and can be used for lead selection, lead optimization, in vivo efficacy screening, and early in vivo toxicological testing. This targeted metabolomics platform can be utilized in the future as a high-throughput screening tool for identification of new biomarker(s) in plasma and CSF of HIV-1 infected individuals.…”

An LC-MS/MS-SRM Method for Simultaneous Quantification of Four Representative Organosulfur Compounds in Garlic Products

“…When considering the specificity, sensitivity, and stability of samples during analysis, liquid chromatography-tandem mass spectrometry-selected reaction monitoring (LC-MS/MS-SRM), which has been used to quantify trace-level analytes in complex matrices, shows promise for the analysis of organosulfur compounds in garlic (Diana Di Mavungu et al 2009;Gianazza et al 2014;Guo et al 2012;Kitteringham et al 2009). Among the organosulfur compounds of garlic, only SAC has been analyzed with LC-MS/MS-SRM to date (Lee et al 2015;Lee et al 2014); the LC-MS/MS-SRM analyses of alliin, GSAC, and allicin, and most importantly, the simultaneous analysis of all four of these garlic compounds have not yet been reported.…”

Metabolômica: Definições, Estado-Da-Arte E Aplicações Representativas

“…49,50 Para aumentar a precisão das quantificações, padrões internos e padrões marcados isotopicamente são comumente utilizados. 51 A análise estatística é usada para extrair a informação relevante dos dados, através de métodos de análise multivariada e univariada. Nas análises multivariadas, a classificação e discriminação de entidades ou metabólitos responsáveis por diferenciar os grupos de amostras são realizadas através da avaliação do conjunto da matriz de dados extraídos na etapa de trabalho anterior.…”