Subsecond absolute quantitation of amine metabolites using isobaric tags for discovery of pathway activation in mammalian cells.

The absolute quantitation of amine metabolites from mammalian cell samples was achieved by combining amine standards, isobaric tags, and capillary liquid chromatography (LC) tandem mass spectrometry (MS/MS). Our approach allowed 32 specific amines to be analyzed within a single chromatographic run, with the generation of the calibration curve and absolute quantitation of each analyte taking less than 900 ms. Using this strategy, we determined the amine response of human aortic endothelial cells (HAECs) from a glucose challenge. The observed changes of the absolute concentration of these metabolites implied eight enzymatic reactions may change efficiency upon glucose treatment. Five of these reactions have been previously reported as being up-regulated in diabetic conditions. The remaining three reactions were analyzed by measuring the expression of these enzymes, with 66% showing increases. Our data indicate that rapid determination of absolute quantitation is useful in determining novel pathway activation. Furthermore, even though we determined the absolute quantity of 32 metabolites here, the number of analytes that can be measured by this method is limited mainly by commercial availability of amine standards.