title: Targeted label-free quantification of interleukin-8 in PMA-activated U937 cell secretome by nanoLC-ESI-MS/MS-sSRM.
abstract: 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.
authors.affiliation: Food Chemistry Unit, Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
authors.forename: Pelin Esma
authors.initials: PE
authors.lastname: Emirbayer
authors.affiliation: Department of Dermatology, Forschungscampus, Universitätsklinikum Erlangen, Erlangen, Germany.|Department of Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
authors.forename: Kerstin F
authors.initials: KF
authors.lastname: Gerer
authors.affiliation: Department of Dermatology, Forschungscampus, Universitätsklinikum Erlangen, Erlangen, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Hoyer
authors.affiliation: Food Chemistry Unit, Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Pischetsrieder
mesh_terms: D002273:Carcinogens / Q000494:pharmacology; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D016209:Interleukin-8 / Q000032:analysis*; D020543:Proteome / Q000187:drug effects / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D020298:U937 Cells
chemical_list: D002273:Carcinogens; D016209:Interleukin-8; D020543:Proteome; D013755:Tetradecanoylphorbol Acetate
keywords: Cytokine quantification; Interleukin-8; Scheduled reaction monitoring; Secretome; Targeted mass spectrometry