85047-08-1Relevant articles and documents
Stable isotope liquid chromatography-tandem mass spectrometry assay for fatty acid amide hydrolase activity
Rakers, Christin,Zoerner, Alexander A.,Engeli, Stefan,Batkai, Sandor,Jordan, Jens,Tsikas, Dimitrios
experimental part, p. 699 - 705 (2012/06/29)
Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d4-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d4-EA) and the internal standard 13C2-EA. Selected reaction monitoring of m/z 66 → m/z 48 (d4-EA) and m/z 64 → m/z 46 (13C2-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d4-AEA hydrolysis obeyed Michaelis-Menten kinetics (KM = 12.3 μM, Vmax = 27.6 nmol/min mg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC50 = 24.3 nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.
Improved CILAT reagents for quantitative proteomics
Zeng, Dexing,Li, Shuwei
supporting information; experimental part, p. 2059 - 2061 (2009/12/03)
Improved CILAT reagents have been developed, with which an unprecedented number of protein samples can be measured in high-throughput assays, providing a robust tool for MS-based quantitative proteomics.