Metabolomics is the study of an organism’s metabolome, which is the collection of metabolites, or small molecules essential for the growth, health, and reproduction of the organism. Differences in metabolomics profiles may reflect different phenotypes or differences in disease state – as such, metabolomics studies are becoming increasingly important in clinical research. There are two main approaches to metabolomics: discovery and targeted.
Metabolomics is complementary to gene expression and proteomic studies. While transcriptomics indicates transcription of DNA into RNA, and proteomics reveals identities and quantities of proteins expressed, metabolomics produces a view into what tasks are performed by proteins - an instant snapshot of the chemistry occurring in the cell. When combined, the integrated data reveals the total biology of the organism, often referred to as integrative biology or systems biology.
Discovery, or untargeted, metabolomics involves a wide-spectrum profiling of all metabolites present in a test group versus a control group. This screening searches for statistically meaningful variations in metabolite abundance between experimental and control groups. Discovery metabolomics may also include identification of previously unknown metabolites, as well as the interpretation of discovered metabolites in the context of biological pathways or conditions. Discovery metabolomics does not require internal standards.
Targeted metabolomics is a directed validation and quantitation of a limited number of metabolites. Targeted assays are built by running chemical standards and developing a library of compounds with known chromatographic retention times, precursor ion masses and product ion masses. Generally these assays are performed using selective reaction monitoring, which focuses the instrument only on the precursor and product ions (transitions) of interest. This allows for the highest possible sensitivity. While known targets MAY be measured during a profiling study, the instrument settings limit the sensitivity of detection.
Relative versus absolute quantitation:
For targeted assays, a reference library is used, with a limited number of reference standards spiked into the sample for quality assurance and sample-to-sample normalization. The MDC goes through pains to establish linear dynamic ranges for all of its assays along with lower limits of detection (LLOD). Therefore, sample relative abundance, and a reportable greater than or less than value may be reported with confidence. This process is referred to as RELATIVE quantitation.
For ABSOLUTE quantitation, heavy internal standards are spiked into the sample in an known quantity. An external standard curve (separate of the samples) is also prepared using light standards, as well as heavy internal standards in the sample type of interest (MATRIX) and in buffer (NO MATRIX control). The ratio of sample to standard abundance is calculated, and this is normalized to the external standard curve for quantitation. While we make standards available at cost, the price may vary, and the mixture of some standards are incompatible (cause suppression or cross-talk). Therefore, absolute quantitation assay pricing may vary widely depending on the target of interest.