Cloud Hosting

Metabolomics is an important discovery tool in numerous research areas, including drug discovery, disease research and crop engineering, as well as in many industrial applications like biomanufacturing. The acquisition of confident measurements depends on accurate metabolite identification and reliable, properly controlled data generation, so an effective methodology is key.High-throughput, large-scale setups are particularly susceptible to variation. So, workflows must be designed with high accuracy and reproducibility in mind to ensure that changes detected are attributable to biology. Recorded spectra are compared to authentic standards for valid compound identification, and multiple quality checks eliminate variation introduced by sample preparation, operator and instrument.Although the focused analysis of a targeted set of metabolites is informative and reliable, sometimes a wider scope is needed to explain a phenotype or explore beyond what is known. Comprehensive metabolome coverage detection of all metabolites in a sample provides that scope but poses high demands on the instrument and setup. First, discriminating the hundreds of metabolites potentially in a sample requires a mass spectrometry (MS) system with high resolution power to tease apart compounds of closely related mass. Second, the instrument must acquire data across multiple ionization modes without bias to fully capture the structural diversity of the metabolome. Third, every run must include calibrations and controls to accurately analyze target metabolites as well as facilitate retro-mining data for unknown molecules.The first two demands are met by modern mass spectrometers that offer exceptional measurement resolution and analytical versatility while the third is met by a well-planned experimental design.

The choice of a control is dictated by the objective of a run. In the case of metabolomic profiling, the internal controls should be a mixture of reference compounds to support identification of target metabolites as well as represent a spectrum of chemical types, molecular weights, concentrations and ion charge. These controls are available commercially as isotope-labeled metabolite mixes figure 2 illustrates the coverage of a commonly used solution.

Figure 2. Spanning the metabolome: internal controls made for metabolomic profiling. Credit: Thermo Fisher ScientificBuilt into a run sequence, internal controls serve two purposes. First, the mix is serially diluted to run as a calibrant to obtain absolute concentration of the sample metabolites. Second, the labelled metabolite mix is spiked into samples at a constant concentration. The retention time, mass accuracy and signal response of each internal standard is then measured throughout the run sequence to assess instrument and method performance.The latter performance evaluation tests for losses or errors in the analytical workflow, ensuring that the instrument and method produce valid data. Steady retention time of the internal control across injections attests to consistent sample processing through separation and measurement, while reproducible mass accuracy and stable signal response verify instrument reliability. As an additional quality check, a pooled sample consisting of aliquots from all test samples is analyzed intermittently throughout the run sequence (typically every 1020 injections). This provides a repeatable measurement of the same sample over the entire sequence or study and confirms a stable signal response for endogenous metabolites.

Based on the cross-run and cross-injection analysis of the internal control summarized in figure 4, the method and HRAM system used performed exceedingly well. Controls from all injections and runs showed minimal deviation in retention time (A), mass accuracy (B) and signal response (C).

By retro-mining the acquired data, urea was found to be at lower concentration in dialyzed FBS (Figure 5B). Uridine, also identified retrospectively by matching data to a spectral library, was more concentrated in heat-inactivated FBS from the US, compared to other US-sourced batches (data not shown).

See the original post here:

The Full Picture, the Right Picture High-Resolution Mass Spectrometry for Metabolomic Profiling - Technology Networks