image   How IROA Works


Isotopic Ratio Outlier Analysis IROA is a simple and direct metabolic profiling technique which embeds unique signatures (tags), specifically using 95% and 5% 13C abundance, into biochemical metabolites for relative quantitation by mass spectrometry (MS) and software algorithms to find, idenitify and quantitate these specific signatures.  IROA can be applied to many life science applications (toxicology, diagnostics, drug development, bioprocess, etc.).

The key to accurate and reproducible identification is to have an internal standard for every metabolite measured.

Why 95% and 5% 13C abundance?  Traditionally, stable isotope labeling analyses use molecules labeled at 99% 13C. The use of 13C isotopes in metabolomics studies greatly enhances the ability to identify and quantify molecules.
Benefits of 95% and 5% 13C labeling:
  • Eliminates false data - 95% and 5% 13C gives rise to a distinctive isotopic pattern easily differentiated from natural abundance non-biological artefacts and noise
  • Mathematically calculable to enable computational analysis
  • Removes variances - labeled and unlabeled species are subject to the same biases, thus increasing comparability of different measurements
  • Enables identification –  the number of carbons for each metabolite can be determined; carbon number and mass together greatly restricts the number of possible molecular formulae 
  • Facilitates detections of low intensity features - at natural abundance more that one isotopic peak is often not detectable or can be excluded as noise
  • Reduces cost of analysis - 95% and 5% 13C samples can be mixed and analyzed together, reducing sample number by half


IROA supplies both pre-labeled complex Internal Standards for targeted or untargeted metabolomics and kits for generating specific Internal Standards taylored to experimental cell types, including bacterial, yeast and mammalian cell lines.

The Basic IROA protocol schematic is presented below.  See also "Phenotypic" analysis where a control or complex "Internal Standard" is lgenerated to spike into experimental samples. 

  1. A homogenous cell population is divided into two populations, a “Control” population and an “Experimental” population (Figure 3-1, blue box).  
    All biological compounds in the Control samples and Experimental samples are labeled with media in which all the carbon sources are randomly and universally composed of approximately 95%12C with 5%13C, and 95%13C with 5%12C, respectively.  The Control and Experimental sample media are chemically identical but isotopically different, and importantly, the isotopes are universally and randomly incorporated into all carbon positions. 
  2. The Control and the Experimental cell samples are grown in the IROA isotopically-defined media for a sufficient time to replace their original natural abundance carbon so that all of their contents will demonstrate distinctive isotopic patterns (orange box).
  3. Once labeled, the Experimental sample is treated with a stressing regimen, and the Control sample is treated with only vehicle (e.g. if drug & DMSO vehicle is added to the Experimental sample, DMSO should be added to the Control sample for the same time period).  The stressing agent may be chemical (e.g. toxin), genetic (e.g. mutant), environmental (e.g. UV exposure), or any element or combination of elements that induce physiological alteration. 
  4. Experimental samples are individually pooled with Control samples, and the resulting composite samples analyzed using LC-MS (Figure 3-1, green box).  This has the effect of reducing sample-to-sample analytical variance, and increasing data quality. 
  5. In the Basic IROA protocol, Control sample metabolic pools are fully labeled with U-95%13C, and Experimental sample metabolic pools labeled with U-5%13C.  Control and Experimental metabolite peaks are easily identified according to the presence of characteristic M-1 and M-1 IROA peak patterns, respectively (see Figure below). 

Figure: Basic IROA signal for Adenosine, C10H12N5O4. The Basic IROA signal is made up of two halves. The C13 side corresponds to the Control metabolite envelope labeled with 95%13C. The C12 side corresponds to the Experimental metabolite envelope labeled with 5%13C. The C12-C13 pairing enables the identity of all peaks. The distance between the two monoisotopic peaks shows this is a 10-carbon compound, which together with accurate mass, is used to name the compound. LC-MS/MS analysis was performed using a Thermo Scientific LTQ Velos mass spec.

The Basic IROA® method grows isotopically labeled cells which are then treated experimentally. When pooled cells are processed the signals for the compounds from both the Control and Experimental cells may be distinguished and differences between the ratio of their areas are directly indicative of the ratio of the respective sizes of their metabolic pools. Outliers to the normalized ratios are metabolic pools that are impacted by the experimental treatment.

The specific signatures in every unique IROA molecule are all mathematically calculable enabling the IROA ClusterFinder™ software tool to easily characterize peaks (as either artifacts, control or experimental compounds), remove artifacts, perform peak correlation analysis to associate adducts with their fragments (new for ClusterFinder2.0), calculate carbon number and molecular formula, quantitate the ratio of control to experimental, and normalize each dataset. Access to the IROA portal for data interpretation and statistical analysis imparts biological meaning to the metabolomics data.