Determination of leukotriene A(4) stabilization by S100A8/A9 proteins using mass spectrometry.
Leukotriene A(4) (LTA(4)) is the precursor for the formation of bioactive leukotrienes, but is highly susceptible to nonenzymatic hydrolysis. Although it is chemically reactive, LTA(4) participates in the process of transcellular metabolism, which requires the transfer of LTA(4) from one cell to another for the production of additional leukotrienes. Due to the susceptibility of LTA(4) to hydrolysis, various methods have been used to measure the half-life of LTA(4) in the presence of different proteins in efforts to understand how it is transported between cells. In this work, a new liquid chromatography mass spectrometry technique was developed to improve upon these previous assays that analyzed LTA(4) directly. The new technique derivatizes LTA(4) to stable compounds for analysis and removes the potential for sample decomposition between analytical runs. This assay was used in measuring the capabilities of the S100A8/A9 protein complex isolated from human neutrophils to stabilize LTA(4). It was determined that the S100A8/A9 protein complex protects LTA(4) from hydrolysis in a Ca(2+) dependent manner and increases LTA(4) half-life to in excess of 35 and 5 min at 4 degrees C and 37 degrees C, respectively.
Rector
Christopher L
CL
Department of Pharmacology, University of Colorado Denver, Aurora, CO 80045, USA.
Murphy
Robert C
RC
D015153:Blotting, Western; D040501:Calgranulin A / Q000737:chemistry / Q000378:metabolism*; D040502:Calgranulin B / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D006801:Humans; D017572:Leukotriene A4 / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D055550:Protein Stability; D053719:Tandem Mass Spectrometry
D040501:Calgranulin A; D040502:Calgranulin B; D017572:Leukotriene A4