XXXX metabolomics of endothelial cells using capillary liquid chromatography mass spectrometry with isotope coded affinity tags.

@entity1 @entity1433 @entity462 @entity4785 @entity2569 @entity2041 @entity2197 @entity1432 @entity73039 @entity1520 @entity1525 @entity413
@entity1432 and @entity1520 levels are critical to maintaining the redox potential of a cell. Perturbations of these levels are important in disease pathogenesis. To improve endogenous @entity1 metabolome quantitation, @entity1432 specific tagging, extraction and relative quantitation were undertaken. Reduced and oxidized @entity1432 ( @entity1520 ) metabolites from endothelial cells were tagged and extracted using cleavable isotope coded affinity tags (cICAT). Extracted cICAT labeled @entity1433 were analyzed using capillary reverse phase liquid chromatography coupled to mass spectrometry (capLC-MS) with positive mode electrospray ionization. Reactions between @entity1432 metabolite standards and the reactive group of cICAT indicate completion by 8h at pH 9 with no apparent @entity1520 formation. cICAT labeled reduced @entity1433 from endothelial cells showed 1-5% RSD using ratiometric quantitation of isotopes and 6-17% RSD based on signal intensity alone. Sample injection was optimized to 16 pmol. Using high mass accuracy MS, 75 putative @entity1432 metabolites were detected in all experimental samples. Treatment of endothelial cells with @entity73039 (BQ) shows decreased levels in 28 putative reduced @entity1433 and increased levels of 27 putative @entity1525 . Treatment of endothelial cells with 30 mM @entity413 resulted in 22 putative reduced @entity1433 with decreased levels and 7 putative @entity1525 with increased concentration. @entity1433 were identified based on accurate mass within 3 ppm and analysis of fragmentation patterns. Using @entity4785 ( @entity4785 ), shared product ions between different @entity1433 led to the analysis of @entity1433 from the @entity462 - @entity2041 ( @entity2569 - @entity2197 ) pathway. Specific reduced @entity1433 and @entity1525 in this pathway revealed changes different from the overall trends of @entity1433 / @entity1525 . This suggests varying regulation of the @entity2569 - @entity2197 pathway distinct from other @entity1432 -containing pathways and dependence on the type of environmental stimulus. These results indicate the utility of analyzing reduced @entity1433 and @entity1525 in eukaryotic samples.
