IROA Biochemical Quantitation Protocol Featured at the Amercian Society for Mass Spectometry

Abstract 1:

Caenorhabditis elegans is one of the best-studied animals in science. Despite this, metabolomic studies in C. elegans have only recently become active areas of research. The Isotopic Ratio Outlier Analysis (IROA) protocol uses 13C-isotopic signatures to identify and to quantitate metabolites (Figure 1). It reduces error introduced during sample preparation and analysis, including ionization suppression by the use of IROA standards. The marriage of IROA and high-resolution accurate mass (HRAM) LC/MS/MS with C. elegans metabolomics allows experiments which assess the biological response to stresses or stimuli. These experiments would conventionally be difficult due to interferences by metabolites of unlabeled organisms. With IROA labeling and HRAM detection, metabolites can be distinguished in an untargeted manner, quantitated and unambiguously identified to their chemical formulas.


Abstract 2:

Myxobacteria represent an important source of novel natural products exhibiting a wide range of biological activities. Some of these so-called secondary metabolites are investigated as potential leads for novel drugs. Traditional approaches to discovering natural products mainly employ bioassays and activity-guided isolation, but genomics-based strategies and “metabolome-mining” approaches become increasingly successful to reveal additional compounds. These newer methods hold great promise for uncovering novel secondary metabolites from myxobacterial strains, as the number of known compounds identified to date is often significantly lower than expected from genome sequence information. Analytical challenges for comprehensive MS-based profiling of myxobacteria include the need to reliably detect the significant differences between secondary metabolomes, e.g. as a consequence of gene knock-outs or regulatory effects, as well as the robust quantitation of known and unknown target compounds and their identification. The IROA protocol was applied to the analysis of myxobacterialsecondary metabolomes.


Abstract 3:

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; becoming 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. 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.