Isotopic ratio outlier analysis (IROA) is a stable isotopic labeling technique that utilizes the creation of distinct signatures in the molecules of a biological sample for identification and quantification. The technique can be used in either an unbiased (untargeted) or targeted metabolic profiling methodology by varying experimental design. The molecular contents of a cell are 'labeled' by replacing their entire carbon content with the stable carbon-13 (13
C) isotope, centered on either a 5% or 95% isotopic balance, in order to create unique, highly informative isotopic patterns. Previous methods of stable isotopic labeling relied on higher purity (typically 98-99% carbon-12) isotopic material which generate very weak (minimal) patterns.
Contents
The use of randomly labeled, uniform isotopic abundance creates isotopic patterns (signatures) of such strength that they are easily detected and used, imparting many analytical advantages to the IROA method. When considering a stable isotope experiment, although many stable isotopes can be considered, it is important to use an isotope that exhibits minimal biological isotopic effect. The advantage of 13
C, which has only an 8% mass difference from 12
C, is that it is generally well tolerated by most cells. One advantage of the IROA protocol over other stable isotopic labeling techniques is that once the labeling step takes place all biologically derived molecules are distinguishable from artifacts (which are present at only natural abundance), and each peak has a signature of its origin, an M+1 for the 12
C sample and an M-1 for the 13
C sample. The mass spectral scans from these pooled samples show the pairing of biological peaks allowing: 1) the removal of artifacts; and 2) the reinforced identification of the compounds the peaks represent because the distance between the paired peaks exactly corresponds to the number of carbons in the molecule.
Protocol
Because all molecules (biochemicals, proteins, RNA and DNA) are labeled by the IROA protocol it can be used in all 'omic sciences, however biochemical profiling or metabolomics is an especially useful case. The natural abundance of carbon is approximately 98.9% 12
C and 1.1% 13
C. Because of this, during mass spectrometric analysis carbon-based molecules have both a monoisotopic peak and a second peak, the "M+1" peak, that is caused by the presence of the isotopes of not only 13
C, but also 17
O, 15
N, 2
H and others. The key to IROA is that analysis is done using a specific mixture of 12
C and 13
C. One isotope is present at approximately 95% and the other at 5%. This the concentrated isotope's corresponding peak dominates the dilute isotope.
Procedure
A homogeneous cell population is divided into equal-sized "experimental" and "control" groups. The biological compounds in these groups are labeled using an isotopically defined growth media in which all of the carbon components in the experimental and control samples are replaced with randomly and universally enriched 5% or 95%13
C, respectively. After at least 5 subsequent cell divisions, the experiment group is exposed to a stressor (chemical, genetic, environmental, etc.) When the experiment has concluded, the experimental sample is mixed with the control sample and analyzed using mass spectrometry. The admixing of samples increases data quality as sample-to-sample variance is reduced and the identification of all biological compounds is enhanced.
ClusterFinder software fully automates the processing of IROA-based data files to identify and quantify all compounds of biological origin and to remove artifacts.
Basic mode
Basic IROA is fundamentally an unbiased analysis. This leads to clean, high resolution data sets that clearly define the biological response of a biological system.
Phenotypic mode
Phenotypic IROA is a targeted analysis for a very large number of compounds. Phenotypic provides a biochemically complex standard against which a biochemically similar natural abundance standard can be compared.
Phenotypic analysis is a diagnostic tool for disease biomarker discovery and drug response in human (or other unlabeled) systems.
Phenotypic is used where it is not possible to isotopically label the patient or biological sample. In that case the sample is collected and mixed with a predefined IROA-labeled "control" or "standard". Since the standard is IROA-labeled, its peaks may be spectrographically identified according to the presence of their characteristic peak. Since the standard is always the same, its spectrograph is known.
The peaks from the samples may be readily identified using ClusterFinder software, because even though they do not carry isotopic labeling, their exact mass and position are established relative to the standard.
Artifacts have no match in the sample and thus need not be considered in a final dataset. With two contrasting IROA populations, a fully targeted metabolomic experiment can be easily and accurately run by running the experimental condition using natural abundance carbon.
Peaks
The basic IROA protocol relies on the creation of isotopic patterns by growing cells on media wherein all the carbon sources contain defined isotopic balances. With reasonable resolution, the mass and the number of carbons will unambiguously indicate a formula for monoisotopic peak. Since this is true for both the 12
C and 13
C peak sets, then either peak carries all of the information needed to find the other (and the formula), the pair of peaks provides a triply redundant mechanism to identify all compounds of biological origin. Since non-biologically derived compounds will never have IROA patterns (like H-NA), all artifactual peaks may be identified and removed from consideration. These characteristics greatly simplify and strengthen the quality for the interpretation of a mass spectrum of a biological sample.
ClusterFinder software
IROA software algorithms were created based on the fact that IROA peaks are mathematically calculable. Each set of carbon isotopomers (12
C and 13
C) reliably and accurately account for the other set, providing a redundant quality control check. The ClusterFinder software achieves a data reduction of complex raw data to concise, high value information by performing these steps: characterization of peaks according to source (artifactual-no label, 12
C experimental, or 13
C control, removal of artifacts, alignment and pairing of remaining peaks across scans, pair normalization and identification, and determination of the relative 12
C/13
C ratios of analytes in each sample.
Applications
The IROA approach is applicable to measuring an organism's biological response to any stressor, such as disease, environment, drugs or toxins. As an example IROA was used to label biosynthetic pathways, specifically outlining the glutathione pathway, during the fermentation process of Saccharomyces cerevisiae (S. cerevisiae). S. cerevisiae was profiled and measured using liquid chromatography/mass spectrometry.