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.

High Resolution Proteomic Analysis of the Cervical Cancer Cell Lines Secretome Documents Deregulation of Multiple Proteases

“…Also, it has been documented as secreted by human hepatocytes (27). In the secretome proteomic analysis, SOD2 was down-regulated in SiHa vs. HCK1T (0.013-fold change, p<0.05) as well as in C33A vs. HCK1T (0.12-fold change, p>0.05), while it was not identified in HeLa cells.…”

A trial proteomics fingerprint analysis of HepaRG cells by FD-LC-MS/MS

“…12 In contrast, the uorogenic derivatization (FD)-LC-MS/MS method 12,13 is a quantitative proteomic analysis method that does not require any sample pretreatment procedure; proteins are derivatized by using uorogenic reagents such as 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) and derivatized proteins are separated by HPLC. This is followed by quantication based on the peak height, fractionation of the protein peaks, enzymatic digestion of the isolated proteins, and the nal identication of the proteins using HPLC and tandem MS. Because this approach has been used for differential proteomics analysis in many biological samples, such as human breast and colorectal cancer cells, and mouse liver, [12][13][14][15] FD-LC-MS/MS should be suitable for a prole proteomics analysis of HepaRG cells. Such analysis should identify the expressed proteins that have been lost during the pretreatment steps of conventional proteomics analysis methods.…”

“…; Imtakt, Kyoto, Japan). 14,15 We investigated the separation efficiency under various ow rates (0.10-0.60 mL min À1 ) (ESI, Fig. 1 †).…”

Συγκριτική Μελέτη Εκκρινόμενων Πρωτεϊνών Από Μια Φυσιολογική Και Καρκινικές Κυτταρικές Σειρές Του Τραχήλου Της Μήτρας Για Την Ανάδειξη Καρκινικών Βιοδεικτών

“…SOD2 επιλέχθηκε για επιβεβαίωση καθώς έχει συνδεθεί και με τον καρκίνο του τραχήλου της μήτρας[57]. Επίσης, έχει μελετηθεί ως εκκρινόμενη σε ανθρώπινα ηπατοκύτταρα[109]. πραγματοποιήθηκε Western blot για την SOD2 στο ολικό κυτταρικό εκχύλισμα, καθώς είναι μια μη κλασικά-εκκρινόμενη πρωτεΐνη και θέλαμε να συγκρίνουμε το πρότυπο έκφρασης της στα κλάσματα του εκκριτώματος και του ολικού κυτταρικού εκχυλίσματος.…”