How the IROA Technology WorksPresentation Transcript
The IROA TM methodology was developed to automate and simplify metabolomics so that investigators can achieve clear and reliable results easily. It only requires suitable isotopically-paired media and software to extract and process the mass spectrometer raw data files. The following slides describe an IROA experiment in its simplest form. This basic methodology may be just as easily applied to simple experimental systems or much more complex experimental systems, only a change in cell medium is required. IROA™: Isotopic Ratio Outlier Analysis
media-free cells Wash Culture Biological A. The use of a pooled biological population at the outset of the experiment will reduce biological variance. The pooled biological population is divided into two and each of these is placed in separate isotopically defined media. B. The control and experimental cells are each allowed to grow through enough divisions to dilute out the natural abundance carbon and replace it with the characteristic isotopic distributions of their particular media. This gives each and every molecule a unique “label”, definitive to source. The experimental conditions are applied to the experimental population, and vehicle to the control population. The IROA™ Analysis 95% 12 C and 5% 13 C media control Experimental Treat Incubate Experimental 95% 13 C and 5% 12 C media drug etc. Add toxin,
C. After the experimentally defined period the experimental and control cells are mixed.
D. The mixed sample, containing both control and experimental populations in one sample, is prepared and analyzed by mass spectrometry as a single sample. The number of samples to be analyzed is reduced by half.
As all of the molecules from each population are distinguishable from one another AND from artifactual molecules, it is possible to look at any peak and understand its origin. In addition, since the control and experimental populations share the same handling-induced variances compound–by-compound (i.e. error or losses), there is no variance between or within them. This “noise-free” environment is a unique feature of IROA TM , and is the most significant reason for its success.
Composite media-free 12 cells + media-free 13 cells Total Ion Data Analytical The IROA™ Analysis
E. Artifacts are discarded; the respective areas for control-derived and experimentally-derived compounds are identified (by isotopic signature) and compared (by ratio).
F. The distribution of ratios is analyzed for outliers. Compounds with abnormal ratios are molecules affected by the experimental stressor, (toxin, drug etc.) allowing for clear interpretation.
The IROA™ Analysis Normalized Ratio 0 -5.0 5.0
IROA Software data flow 10 8 datapoints (mostly noise) B. C. A. 5X10 2 datapoints (pure data) Variance control, Data reduction, Noise removal, Data definition Fully automated data reduction of complex raw data to concise, high value information. The IROA TM software is capable of interpreting these datasets, and will allow for a very dramatic reduction in data size as it easily sorts through the dataset removing irrelevant data. No other system can do this as efficiently or consistently. Normalized Ratio 0 -5.0 5.0