The IROA Phenotypic Experiment (only the control is IROA-labeled)

In the typical IROA experiment, both control and experimental samples are labeled in-vitro. There are many cases where it is not practical or possible to label the experimental sample, for example, when working with tissue samples, performing large fermentation runs, or propagating field-grown plants. In these instances, the IROA “Phenotypic” experiment may be applied. In the Phenotypic IROA experiment, the control or “Standard” is isotopically labeled using IROA C13 media. An ideal Standard is one that represents the entire metabolome of the fluid or tissue under study. For example, if interrogating the metabolome in pancreatic tissue, quantification of metabolites can be achieved using an IROA-grown pancreatic cell line as a complex internal Standard. For Phenotypic analyses, the ClusterFinder software will find all IROA-labeled compounds where the IROA peak is present in one sample but not the other. This allows for a complex targeted analysis whereby the 13C IROA peaks in the labeled control sample are used to identify their associated natural abundance peaks in the experimental sample.

The IROA Phenotypic Experiment. The material to be phenotyped, a breast biopsy, is mixed with 13C IROA-Standard compounds which is used by the ClusterFinder software to find and pair all peaks. For each metabolite, the deviation from the paired metabolite in the Standard is diagnostic of the phenotype of the sample.

Using an IROA-labeled Standard, all of peaks may be easily identified according to presence of their characteristic M-1 peak. The natural abundance peaks corresponding to each Standard peak may be readily identified because even though they do not carry any isotopic labeling, their exact mass and position are established relative to the Standard. If the pooled Standard is well characterized and the compounds that are present in it have already been identified, then it will be able to be used as a point of comparison for all of its contained compounds. Artifacts will have no match in the Standard and are eliminated by ClusterFinder in a final dataset. In a typical experimental IROA dataset the ratio of the peak areas represent the relative deviation of the metabolic pool sizes brought about by the experimental condition. In a Phenotypic experiment, all experimental samples are measured relative to the Standard; therefore the phenotype is defined by the deviation from the Standard.

The Phenotypic IROA signal is made up of two halves.  The C13 side tells the ClusterFinder software where to find the corresponding 12C peak.  The C12 side is unlabeled and has only natural abundance carbon.  The pairing makes the identity of all peaks measured unambiguous. 

View the Application Note: Differential Metabolomic Profiling of Maize Genotypes under Drought-Stressed Conditions using IROA® (Isotopic Ratio Outlier Analysis)

  A tutorial on the Phenotypic Experiment can be viewed here.