Systems biology has emerged in the previous decade to provide a holistic study of the biochemical components (genes, transcripts, proteins, and metabolites) and their complex interactions that create the emergent properties or phenotype of biological systems. Metabolomics is a core discipline of systems biology and involves the investigation of low molecular weight organic and inorganic metabolites present in a cell, tissue, organ, or organism.
Metabolomics provides a dynamic and representative phenotypic picture of the system involving endogeneous and exogeneous metabolism and biochemical regulation (e.g., allosterism and riboswitches).
Metabolomic studies are a multistage process involving hypothesis-generation discovery and validation studies. The workflows for experiments and metabolite identification are shown in Figures 1 and 2. Many metabolomics studies start from a point of limited biological knowledge.
A holistic experiment is designed and performed to acquire robust and valid data encompassing a wide and diverse range of metabolites and metabolic pathways. Data is interrogated to define metabolic differences between classes, for example, two classes of subjects diagnosed/not diagnosed with a disease. This strategy is defined as metabolic profiling.
Experimental design is essential to ensure biases (for example, age, gender, drugs) between classes being studied are not present and the use of quality control samples is highly recommended to allow the quantitation of technical and biological variation.
The human metabolome is estimated to contain greater than 7,800 metabolites although many metabolites related to drug, lipid, and gut microflora metabolism are currently not accurately described. Powerful analytical technologies are required to fulfill the objectives of metabolic profiling. Chromatography-mass spectrometry platforms are routinely applied in metabolic profiling, including ultra high performance liquid chromatography (UHPLC) coupled to hybrid mass spectrometry instruments. These offer reproducible detection of thousands of features from a given sample due to advanced chromatographic and mass resolution.
The power of UHPLC coupled to the hybrid mass spectrometer will be described in this article. Specifically the application of the Thermo Scientific LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific), coupled with UHPLC, to the role of chemical identification of metabolites will be discussed.