ABSTRACT
There are two important processes in sample preparation: quenching and extraction. Quenching is the process of stopping biological reactions in a cell, and extraction is the process of obtaining metabolites from the cell. Sample quenching has been focused on stopping metabolism at a specific period to measure the true quantity of metabolites at a given time. For quenching, the following are required to measure the quantity of metabolite in a cell accurately: (1) a short timeframe during which the biological reaction is stopped, and (2) limited leakage of metabolite and reproducibility. The appropriate extraction method must be chosen considering the following: (1) cell properties, such as robustness of the cell membrane, (2) chemical properties of the target analyte, and (3) reactivity of enzymes (Putri et al., 2013). In this chapter, we introduce the sample preparation protocol for metabolomics using various specimens namely microbial, plant, animal, medical, and food samples. (See Figure 3.1 and Table 3.1.)
The microbe is an important sample for the field of metabolomics because it has been used for the development of experimental procedures and construction of research tactics
(Putri et al., 2013). Quantitative understanding of microbial metabolism and in vivo regulation entails comprehensive coverage of both extracellular and intracellular metabolites. There are two approaches for quantification in metabolomics, namely relative and absolute quantification. Relative quantification normalizes the metabolite signal intensity to that of an internal standard or another metabolite and is typically used in nontargeted large-scale profiling. Absolute quantification uses external standards or internal isotopically labeled standards to determine the absolute metabolite quantity and is mostly used in targeted metabolomics (Lei, Huhman, and Sumner, 2011). In general, the experimental procedures in microbial metabolomics include: (1) biomass cultivation, (2) fast sampling and instant arrest of metabolic activity and deactivation of endogenous enzymatic activity (also widely known as quenching), (3) metabolite extraction, and (4) subsequent quantification of intracellular reactants (metabolites; Mashego et al., 2007; Winder et al., 2008).
