title: Comprehensive and temporal analysis of secreted proteins in the medium from IL-6 exposed human hepatocyte.
abstract: We have previously identified intracellular secretory acute phase response (sAPR) proteins in human hepatocytes following interleukin-6 (IL-6) induction by fluorogenic derivatization (FD)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS). In this report, we utilized this method, which uses 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as the FD reagent, to comprehensively and time-dependently analyze secreted proteins in the medium, including sAPR proteins. Since DAABD-Cl selectively reacts with thiol moieties of cysteinyl residues, direct derivatization, high-resolution LC separation and identification of the secreted proteins in the culture medium were successfully achieved without a pretreatment step. As a result, 14 sAPR proteins were identified simultaneously during a 72 h induction by IL-6. The secretion levels of 11 proteins increased, whereas the secretion levels of three important transport proteins decreased (albumin, retinol-binding protein 4 and transthyretin). In addition, the secretion level of a haptoglobin was found to increase significantly between 0 and 6 h by 1.88-fold compared with the control sample. The secretion levels of four cytoplasmic proteins increased: LDH, a known marker for cell damage, and GSTA1, FABP1 and ADH1B, which are marker proteins for hepatocellular damage. The secretion levels of the other two newly identified cytoplasmic proteins, profilin-1 and SOD2, were also found to increase, suggesting that these two proteins represent novel markers for cell damage. These results suggest that the FD-LC-MS/MS proteomics method can be used to analyze comprehensively and time-dependently the secreted proteins and thereby can offer information that aids our understanding of the dynamics of protein secretion affected by the exposure of cytokines such as IL-6.
authors.affiliation: Laboratory of Proteomics Analysis, Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo, Tokyo, 202-8585, Japan; Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka, 412-8513, Japan.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Nakata
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Saitoh
authors.forename: Jun
authors.initials: J
authors.lastname: Amano
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ichibangase
authors.forename: Masaki
authors.initials: M
authors.lastname: Ishigai
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Imai
mesh_terms: D002851:Chromatography, High Pressure Liquid; D022781:Hepatocytes / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D015850:Interleukin-6; D011506:Proteins
keywords: FD-LC-MS/MS method; Interleukin-6; human hepatocyte; secreted proteins