Targeted label-free quantification of interleukin-8 in PMA-activated U937 cell secretome by nanoLC-ESI-MS/MS-sSRM.

Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12-myristate 13-acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin-8 (IL-8). Next, a label-free nanoLC-ESI-MS/MS-sSRM method for quantification of IL-8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM-MS using one proteotypic peptide as precursor ion and four mass transitions. Label-free quantification was performed by external calibration using IL-8 standard. Validation results indicated that the method was suited for the quantification of IL-8 in the secretome. The maximal IL-8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL-8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.

Proteomic Response of Human Umbilical Vein Endothelial Cells to Histamine Stimulation.

The histamine receptors (HRs) represent a subclass of G protein-coupled receptors (GPCRs) and comprise four subtypes. Due to their numerous physiological and pathological effects, HRs are popular drug targets for the treatment of allergic reactions or the regulation of gastric acid secretion. Hence, an understanding of the functional selectivity of HR ligands has gained importance. These ligands can bind to specific GPCRs and selectively activate defined pathways. Supporting the activation of a therapeutically necessary pathway without the activation of other signaling cascades can result in drugs with more specific activity and fewer side effects. To evaluate the cellular consequences resulting from receptor binding, comprehensive analyses of cellular protein alterations upon incubation with ligands are required. For this purpose, endothelial cells are treated with histamine, as the endogenous ligand of HRs, to obtain a global overview of its cellular effects. Quantitative proteomics and pathway analyses of histamine-treated and untreated cells reveal enrichment of the nuclear factor-κB and tumor necrosis factor signaling pathways, cytokine-cytokine receptor interactions, complement and coagulation cascades, and acute inflammatory processes upon histamine treatment. This strategy offers the opportunity to monitor HR-mediated signaling in a multidimensional manner.