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.