This core will provide a hybrid (targeted, non-targeted) metabolomics platform to support chemical profiling of RBC and other biological samples as needed by the POI studies. The central platform consists of dual chromatography-high-resolution mass spectrometry coupled with a series of data extraction algorithms that provide relative quantification of up to 90,000 high-mass accuracy (5 ppm) m/z features, representing in excess of 10,000 chemicals present in human samples. Targeted analysis allows direct measurement of over 300 chemicals with known identity and capabilities to detect unidentified and unknown chemicals contributing to outcomes. Experience with analyses of thousands of samples shows that the method measures ions that match more than half of the metabolites in human metabolic charts; selected confirmation of identities by co-elution and MS/MS shows that the approach provides an effective global survey of metabolism, capturing metabolites in 146 out of 154 pathways in KEGG human metabolic pathways. Potentially useful for the current proposal, the approach also detects thousands of chemicals derived from diet, the host microbiome, infectious agents and environmental exposures, which could contribute to variation in recipient susceptibility to transfusions of stored RBCs. Rigorous adherence to standard operating procedures using dedicated instruments with internal standards and external pooled reference samples will provide the capability to support metabolome-wide association studies (MWAS) to identify both endogenous and foreign chemicals in RBCs that are associated with specific clinical and/or functional outcomes in the proposed studies. Samples are analyzed in triplicate so that coefficients of variation are determined for each m/z feature in each sample. Selection of data extraction parameters allows more liberal data extraction criteria for discovery-based analysis and more rigorous targeted criteria using stable isotope dilution and multiple ion monitoring to test specific hypotheses. For unknown and unidentified chemicals, mass spectral data consisting of accurate mass m/z, retention time and relative quantification as ion intensity are used with data-based searching for putative chemical identification. Data dependent ion-dissociation (MS/MS and MS) analyses are provided on representative or selected samples to confirm identities of targeted metabolites and/or high abundance m/z features.
The success of this Program is predicated on the ability to perform rapid, cost-effective metabolomic analyses, and correlate changes in specific metabolites with selected important clinical outcomes (RBC survival, vascular inhibition, necrotizing enterocolitis, and GvHD). This Metabolomics Core is critically to achieving this objective.
Showing the most recent 10 out of 72 publications
