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Comprehensive and temporal analysis of secreted proteins in the medium from IL‐6 exposed human hepatocyte
Author(s) -
Nakata Katsunori,
Saitoh Ryoichi,
Amano Jun,
Ichibangase Tomoko,
Ishigai Masaki,
Imai Kazuhiro
Publication year - 2014
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3103
Subject(s) - chemistry , secretion , derivatization , secretory protein , transthyretin , acute phase protein , biochemistry , blood proteins , albumin , hepatocyte , chromatography , mass spectrometry , biology , in vitro , immunology , inflammation , endocrinology
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. Copyright © 2013 John Wiley & Sons, Ltd.