A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein.

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

A proteomics profile analysis was performed on a human hepatocyte carcinoma cell line (HepaRG) by using the FD-LC-MS/MS method. One hundred and fifty-eight proteins were newly identified for the first time of which 10 were found to be specific to human hepatocytes. These proteins are a "proteomics fingerprint" that can be used to characterize HepaRG cells.

Comprehensive and temporal analysis of secreted proteins in the medium from IL-6 exposed human hepatocyte.

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.

Improved separation of fluorogenic derivatized intact proteins with high resolution and efficiency using a reversed-phase liquid chromatographic system.

Although the efficient separation of intact protein mixtures is extremely difficult, reversed-phase chromatography is an important technique for performing quantitative, accurate and reproducible proteomics analyses. Here, we show that, despite the operating constraints of conventional high-performance liquid chromatography, such as column temperature, operating pressure and separation time, comprehensive separation of fluorogenic derivatized intact proteins could be achieved with high resolution and separation efficiency. First, amylin was chosen as a model peptide and used to estimate the separation efficiency with respect to column temperature and flow rate, as indicated by peak capacity. Then, an extract of human primary hepatocytes was used to model complex component mixtures and the separation conditions were optimized. The effects of mobile-phase pH, the separation time and the column length were also investigated. Consequently, more than 890 peaks could be separated efficiently in the extract, which is 1.5-fold greater than when using conventional conditions. Finally, it was demonstrated that both longer separation time and column length contributed greatly to the effective separation of the protein mixture. These results are expected to provide insights into the separation of intact proteins.

Long-term follow-up outcome of imatinib mesylate treatment for recurrent and unresectable gastrointestinal stromal tumors.

BACKGROUND: The follow-up study of up to 71 months of a randomized phase II B2222 trial has demonstrated a long-term survival in patients with recurrent or unresectable gastrointestinal stromal tumors (GISTs). One subset of the patients (17.7%) has been alive for over 9 years with continuous imatinib mesylate (imatinib) treatment. Here, we report the retrospective analysis of recurrent or unresectable GIST patients with imatinib treatment at our institution. METHODS: We summarized the data of 20 patients with recurrent or unresectable GIST treated with imatinib. RESULTS: Patients were followed for a median of 40 months (range 2.5-103) under imatinib treatment. The median progression-free survival (PFS) was 89 months and overall survival for 8 years was 67%. Fifteen patients showed continuous partial response or stable disease with imatinib treatment. The median PFS was 45 months and the median size of the primary tumor was 7.6 cm (range 2.8-18). Four patients showed progressive disease. The median PFS was 56 months and the median size of the primary tumor was 11.9 cm (range 6.7-19). Grade 3 or 4 adverse events occurred in neutropenia (10%), anemia (15%) and renal dysfunction (5%). However, all patients were well managed by supportive treatment and none were discontinued from imatinib treatment due to toxicity or adverse events. CONCLUSION: Imatinib had a high efficacy in patients with unresectable and recurrent GIST during long-term follow-up. All patients were well managed by supportive treatment against adverse events and they were able to take imatinib without discontinuation. The management of adverse events was a key factor for achieving a long-term survival. In addition, the potential risk of imatinib-resistant GISTs tends to depend on the size of the primary GISTs.

Alteration of intracellular secretory acute phase response proteins expressed in human hepatocyte induced by exposure with interleukin-6.

Interleukin-6 (IL-6) is a principal proinflammatory cytokine inducing the acute phase response in various tissues, including liver. Here, we adopt the FD-LC-MS/MS method, consisting of fluorogenic derivatization (FD), separation by liquid chromatography (LC), and identification of proteins by LC-tandem mass spectrometry (MS/MS), to reveal how exposure to IL-6 alters temporally the intracellular secretory acute phase response (sAPR) proteins expressed in human hepatocytes as compared to non-exposure. Nine altered sAPR proteins were identified in cultures in response to IL-6. Seven of them (serum amyloid A protein, haptoglobin, fibrinogen α chain, fibrinogen ß chain, fibrinogen γ chain, α(1)-acid glycoprotein and α(1)-antitrypsin) were significantly increased and two (ß(2)-glycoprotein 1 and transferrin) were significantly decreased in response to IL-6. In addition, the transmission speed of transferrin might be much faster than the other sAPR proteins. These results suggest a different molecular mechanism for protein synthesis and the secretory pathway among the sAPR proteins. In this study, we observed the simultaneously and temporally altered expression of sAPR proteins which had been induced by exposure to IL-6 in human hepatocytes, in contrast to previous reports, in all of which the proteins were tested from the time they were secreted into the medium from the cells.

Towards clinical proteomics analysis.

To understand accurate protein dynamics, a highly reproducible proteomics analytical method is required. The acquired thus knowledge will lead to the diagnosis, treatment and protection against diseases. This review deals with proteomics analysis from a view of sample pre-treatment, sensitivity and reproducibility of the methods, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), liquid chromatography-tandem mass spectrometry (LC-MS/MS). and fluorogenic derivatization (FD)-LC-MS/MS.

A ubiquitin E2 variant protein acts in axon termination and synaptogenesis in Caenorhabditis elegans.

In the developing nervous system, cohorts of events regulate the precise patterning of axons and formation of synapses between presynaptic neurons and their targets. The conserved PHR proteins play important roles in many aspects of axon and synapse development from C. elegans to mammals. The PHR proteins act as E3 ubiquitin ligases for the dual-leucine-zipper-bearing MAP kinase kinase kinase (DLK MAPKKK) to regulate the signal transduction cascade. In C. elegans, loss-of-function of the PHR protein RPM-1 (Regulator of Presynaptic Morphology-1) results in fewer synapses, disorganized presynaptic architecture, and axon overextension. Inactivation of the DLK-1 pathway suppresses these defects. By characterizing additional genetic suppressors of rpm-1, we present here a new member of the DLK-1 pathway, UEV-3, an E2 ubiquitin-conjugating enzyme variant. We show that uev-3 acts cell autonomously in neurons, despite its ubiquitous expression. Our genetic epistasis analysis supports a conclusion that uev-3 acts downstream of the MAPKK mkk-4 and upstream of the MAPKAPK mak-2. UEV-3 can interact with the p38 MAPK PMK-3. We postulate that UEV-3 may provide additional specificity in the DLK-1 pathway by contributing to activation of PMK-3 or limiting the substrates accessible to PMK-3.

Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.

Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. We report that RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1(lf) mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. Our data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade.