Tomoko Ichibangase

A proteomics study on human breast cancer cell lines by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry.

Although several molecular markers for human breast cancer exist, their versatility is limited. Here we demonstrate, through a differential proteome analysis utilizing the fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) method between seven cancer cells and one normal cell, that the presence of cooperatively expressed annexin-2 and galectin-1 without tropomyosin-1 in a tissue could be used to diagnose metastatic breast cancer. Interestingly, in a metastatic cancer cell, the expression of the former two together with highly expressed cofilin-1 activates the Rho signal pathway to aggressively form disorganized actin filaments. Despite the excess expression of annexin-2 and galectin-1 in the normal cell, the highly expressed tropomyosin-1 counteracted the activity of cofilin-1 and stabilized the filaments, resulting in the restoration of the disorganization. This phenomenon suggests that enhancement of tropomyosin-1 should be used as therapy for metastatic breast cancer.

Limitation of immunoaffinity column for the removal of abundant proteins from plasma in quantitative plasma proteomics.

In plasma proteomics, before a proteome analysis, it is essential to prepare protein samples without high-abundance proteins, including albumin, via specific preparation techniques, such as immunoaffinity capture. However, our preliminary experiments suggested that functional changes with use alter the ability of the immunoaffinity column. Thus, in this study, to evaluate the changes of the removal ability of abundant proteins from plasma by the immunoaffinity column, plasma proteome analysis was performed for the long-term test for the reproducibility of the affinity column using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method combined with an IgY column. The specific adsorption for albumin decreased with an increase in the number of the column usage before its expiration date. Moreover, it was demonstrated that hydrophobic high molecular weight compounds in plasma adsorbed onto the column materials surface contributed to the functional changes from specific immunoaffinity adsorption into hydrophobic interaction. These results suggested that, in quantitative plasma proteomics studies, it is important to keep in mind the risk of not only the nonselective loss but also the changes in the adsorption ability of the immunoafinity column.

Comprehensive fluorogenic derivatization–liquid chromatography/tandem mass spectrometry proteomic analysis of colorectal cancer cell to identify biomarker candidate

Existing colorectal cancer biomarkers are insufficient for providing a quick and accurate diagnosis, which is critical for a good prognosis. More appropriate biomarkers are thus needed. To identify new colorectal cancer biomarker candidates, we conducted a comprehensive differential proteomic analysis of six cancer cell lines and a normal cell line, utilizing a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) approach. Two sets of intracellular biomarker candidates were identified: one for colorectal cancer, and the other for metastatic colorectal cancer. Our results suggest that cooperative expression of FABP5 and cyclophilin A might be linked to Her2 signaling. Upregulation of LDHB and downregulation of GAPDH suggest the existence of a specific nonglycolytic energy production pathway in metastatic colorectal cancer cells. Downregulation of 14-3-3ζ/δ, cystatin-B, Ran and thioredoxin could be a result of their secretion, which then stimulates metastasis via activity in the sera and ascitic fluids. We propose a possible flow scheme to describe the dynamics of protein expression in colorectal cancer cells leading to tumor progression and metastasis via cell proliferation, angiogenesis, disorganization of actin filaments and epithelial-mesenchymal transition. Our results suggest that colorectal tumor progression may be regulated by signaling mediated by Her2, hypoxia, and TGFβ.

Application of Fluorogenic Derivatization-Liquid Chromatography-Tandem Mass Spectrometric Proteome Method to Skeletal Muscle Proteins in Fast Thoroughbred Horses

To extend the applicability of the fluorogenic derivatization-high performance liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method, which consists of fluorogenic derivatization (FD), separation by liquid chromatography (LC), and identification by LC-tandem mass spectrometric (MS/MS) proteomic analysis, we applied it to Thoroughbred horse muscle. With the optimization of the protein extraction and separation procedure, reproducible chromatograms were obtained and the changes in protein expressions during exercise were able to be analyzed. To quantify the changed protein expressions, the training-to-detraining (+/-) ratios for proteins were calculated, and the correlation of the ratio with the percentage of maximum oxygen consumptions (VO2max; the indicator of the running speed) was investigated. Sixteen proteins involved in energy supply, especially in anaerobic energy production, increased with an increase in VO2max, suggesting that this method was able to suggest the biochemical events in the faster-running horse and would be useful for evaluating the training effect in Thoroughbred horses.

Application of an improved proteomics method, fluorogenic derivatization–liquid chromatography–tandem mass spectrometry, to differential analysis of proteins in small regions of mouse brain

To identify age-related proteins in small regions of mouse brain, we improved a proteomics approach, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and applied the method to the differential proteome analysis of aging in cerebral cortex, hippocampus and brainstem. The method showed good accuracy with RSDs <10% for between-day protein peak heights, and much better sensitivity for the detection of proteins compared to other proteomics approaches. The existence of 28 regionally specific age-related proteins in mouse brain was demonstrated. These results verified that the small brain regions could be the targets for proteome analysis by the FD-LC-MS/MS method.

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 β chain, fibrinogen γ chain, α(1)-acid glycoprotein and α(1)-antitrypsin) were significantly increased and two (β(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.

A toxicoproteomic study on cardioprotective effects of pre-administration of docetaxel in a mouse model of adriamycin-induced cardiotoxicity

Studies suggest that pre-administration of docetaxel (DOC) in adriamycin (ADR)-DOC combination anticancer therapy results in stronger antitumor effects and fewer ADR-induced cardiotoxic deaths in mouse model, yet no mechanism explaining this effect has been established. The aim of this study was to identify cellular processes in mouse heart tissue affected by different ADR/DOC dosing protocols using a toxicoproteomic approach. We applied fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) - which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry - to the proteomic analysis of heart tissue from control, intermittent-dosing (DOC-ADR), and simultaneous-dosing (ADR&DOC) groups. In DOC-ADR group, ADR was administered 12h after DOC injection; in ADR&DOC group, both drugs were administered simultaneously; in control group, saline was administered at the same timing as ADR injection of other groups. Heart samples were isolated from all mice 1 week after the treatment. The highly reproducible and sensitive method (FD-LC-MS/MS) identified nine proteins that were differentially expressed in heart tissue of control and the two treatment groups; seven of these nine proteins participate in cellular energy production pathways, including glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain. Significantly higher expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was observed in the DOC-ADR group, the group with the fewer cardiotoxic deaths, than in the ADR&DOC group. Therefore, GAPDH may have potential as a drug target for protective intervention and a biomarker for evaluation of the cardioprotective effects in pre-clinical studies.

Liquid chromatographic separation of proteins derivatized with a fluorogenic reagent at cysteinyl residues on a non-porous column for differential proteomics analysis.

A wide-pore (30 nm) reversed-phase column (Intrada WP-RP, particle size 3 μm) was recently utilized for protein separation in differential proteomics analysis with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and exerted a tremendous effect on finding biomarkers (e.g., for breast cancer). Further high-performance separation is required for highly complex protein mixtures. A recently prepared non-porous small-particle reversed-phase column (Presto FF-C18, particle size: 2 μm) was expected to more effectively separate derivatized protein mixtures than the wide-pore column. A preliminary experiment demonstrated that the peak capacity of the former was threefold greater than that of the latter in gradient elution of a fluorogenic derivatized model peptide, calcitonin. The FD-LC-MS/MS method with a non-porous column was then optimized and applied to separate liver mitochondrial proteins that were not efficiently separated with the wide-pore column. As a result, high-performance separation of mitochondrial proteins was accomplished, and differential proteomics analysis of liver mitochondrial proteins in a hepatitis-infected mouse model was achieved using the FD-LC-MS/MS method with the non-porous column. This result suggests the non-porous small-particle column as a replacement for the wide-pore column for differential proteomics analysis in the FD-LC-MS/MS method.

Evaluation of lophine derivatives as L-012 (luminol analog)-dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2.

8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L-012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine-based CL enhancers of the horseradish peroxidase (HRP)-catalyzed CL oxidation of luminol, namely 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI), 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), and 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L-012 and evaluated these as L-012-dependent CL enhancers. In addition, to detect HRP and/or H2O2 with higher sensitivity, each detection condition for the L-012-HRP-H2O2 enhanced CL was optimized. All the derivatives enhanced the L-012-dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L-012-dependent CL using 4-iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H2O2 detection, the detection limits for enhanced CL with HPI and 4-iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L-012-dependent CL enhancer.

Toxicoproteomic analysis of a mouse model of nonsteroidal anti-inflammatory drug-induced gastric ulcers.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents; however, their use has been limited by their association with mucosal damage in the upper gastrointestinal tract. NSAIDs inhibit cyclooxygenase and consequently block the synthesis of prostaglandins, which have cytoprotective effects in gastric mucosa; these effects on prostaglandins have been thought to be major cause of NSAID-induced ulceration. However, studies indicate that additional NSAID-related mechanisms are involved in formation of gastric lesions. Here, we used a toxicoproteomic approach to understand cellular processes that are affected by NSAIDs in mouse stomach tissue during ulcer formation. We used fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS)-which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry-in this proteomic analysis of pyrolic stomach from control and diclofenac (Dic)-treated mice. FD-LC-MS/MS results were highly sensitive; 10 differentially expressed proteins were identified, and all 10 were more highly expressed in Dic-treated mice than in control mice. Specifically, expression levels of 78 kDa glucose-regulated protein (GRP78), heat shock protein beta-1 (HSP27), and gastrin were more than 3-fold higher in Dic-treated mice than in control mice. This study represents a first step to ascertain the precise actors of early NSAID-induced ulceration.