The IROA protocols can be applied to a wide range of Life Science and Diagnostic disciplines and utilize tools that help researchers achieve more accurate metabolic data analysis. This section provides examples of Application Notes and Whitepapers using the IROA protocols including metabolic profiling and the TruQuant isotopically labeled yeast workflow using different vendor instrumentation (Thermo, Sciex, Bruker).
Application Notes and Whitepapers
Isotope Ratio Outlier Analysis (IROA) and Variable Window SWATH® Acquisition allows for Unambiguous Metabolite Identification
This Application Note applies a variable window SWATH acquisition strategy to the data analysis of an IROA Internal Standard (IS) spiked-sample to make it possible to unambiguously identify and accurately quantify hundreds of detectable metabolites in a single unbiased metabolomics analysis using ClusterFinder Software.
The IS contains 500+ well characterized metabolites, which migrated in an HPLC separation with their natural abundance isotopomers, and enabled both identification and standard quantitation for accurate measurement even in a non-baseline, “unbiased” metabolomics separation. Using traditional DIA, all compounds with the same retention time are fragmented without selection, minimal benefit is gained and the process is manually work intensive.
HepG2 Labeling and Cell Growth Protocol for IROA Phenotypic Metabolic Profiling
The IROA protocol for metabolic profiling utilizes full metabolic labeling to distinguish between biological compounds (compounds that arise from metabolic networks found in control and experimental samples) and noise and chemical artifacts such as plasticizers, airborne and waterborne contaminants.
Where it is not possible to label the biological sample, the “Phenotypic” IROA Protocol is applied. Here the experimental sample is collected at natural abundance and mixed with a fully predefined “Standard” that has been isotopically labeled using IROA media in which all of the carbon components are randomly labeled at 95% U-13C. An ideal Standard would be one that represented the entire metabolome of the sample under study. This Application Note describes the labeling and cell growth of HepG2 for use as an internal Standard.
The IROA protocol has been applied in a phenotypic analysis of field grown maize (Zea mays) to understand the biochemical differences across selected genotypes when exposed to drought conditions. In this IROA phenotypic analysis, field-grown leaves containing carbon at natural abundance were compared to a standard maize leaf that was grown to contain universally-distributed ~97% 13C; resulting in a targeted analysis using a biologically-relevant internal standard. At 97% 13C the IROA patterns were sufficient to find isotopically labeled peaks, identify their 12C isotopomers, and remove artifacts, noise and extraneous peaks using the IROA ClusterFinder software.
With accurate mass and IROA, the identification of observed component peaks to chemical formula is unambiguous. The benefit of IROA is it takes into account variances introduced during sample-preparation and analysis, including ion suppression.
Myxobacteria represent an important source of novel natural products exhibiting a wide range of biological activities. In this Application Note, the IROA tools coupled with the Bruker Daltonik UHR- Q-TOF instrument (maXis 4G) were employed to analyze the secondary metabolome of myxobacteria to detect changes triggered by differential iron supply.
Caenorhabditis elegans is one of the best-studied animals in science and the genetics of this “worm” is especially well defined. This Application Note describes the quantitation and identification of C. elegan metaboliites using the IROA protocol and Thermo Scientific HR/AM Q Exactive LC-MS/MS.