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Publications (7)13.57 Total impact

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    ABSTRACT: We previously reported peptide candidates of disease biomarkers (DBMs) for pregnancy-induced hypertension syndrome (PIH) using a novel peptidomic analytical method, BLOTCHIP(®)-MS. The aim of this study was to establish a sandwich enzyme-linked immunosorbent assays (ELISA) system for quantitation of such peptides and to validate their usefulness as DBMs of PIH including gestational hypertension/preeclampsia (GH/PE). We focused on three peptide fragments, kininogen-14 3 8 - 4 5 6 (PDA039), kininogen-14 3 9 - 4 5 6 (PDA044) and cysteinyl α2-HS-glycoprotein3 4 1 - 3 6 7 (PDA071). Using polyclonal antibodies (pAbs) specific for each peptide, suitable conditions for the sandwich ELISA system were investigated. The quantitative ELISA values were confirmed by quantitative matrix assisted laser desorption/ionization time-of-flight MS analyses. Using the established ELISA systems, sera from GH/PE patients and paired serum samples from healthy pregnant females were analyzed. The optimum sandwich ELISA conditions for PDA039/044 quantitation were developed. Quantitation of PDA071 by ELISA failed, presumably due to issues with pAb specificity for the native peptide. Bland-Altman plots showed a satisfactory correlation between the serum PDA039/044 concentration by ELISA and that by quantitative MS analysis. Although the PDA044 level showed no significant change during pregnancy, including in GH/PE patients, the serum PDA039 level was significantly increased (P < 0.0001) in the patients. The simple quantitation technology for PDA039 by ELISA was established for the first time. PDA039 is confirmed its clinical utility as a DBM for GH/PE by the ELISA system using clinical samples. The information provided from present study would be new valuable addition in the field of GH/PE research. © 2015 Sage Publications, Inc.
    Annals of Clinical Biochemistry 04/2015; DOI:10.1177/0004563215583697 · 2.08 Impact Factor
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    ABSTRACT: Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide. Early CRC diagnosis is critical, since patients diagnosed at an early stage have an increased five-year survival rate after surgical resection. Serum biomarkers for CRC detection have been described and peptidomic analysis is a promising approach because of its diagnostic potential. A total of 72 CRC patients and 63 healthy controls were investigated. We used a comprehensive peptide analysis technique, BLOTCHIP®-MS analysis, a combination of electrophoresis and mass spectrometry, for high sensitivity detection of trace amounts of serum peptides. The prediction model comprised five peptides: m/z 1616.66 (fibrinogen alpha chain), m/z 2390.26 (alpha-1-antitrypsin), m/z 2858.42 (AHSG S-cysteinylated form), m/z 3622.78 (VASP), and m/z 3949.98 (F. XIIIa). The three CRC groups, stages II to IV, II + IIIa, and IIIb + IV, were discriminated from controls. High diagnostic performance was suggested by AUC (0.924), sensitivity (83%), specificity (92%), and median probability ratio (6.80) to CRC stage II to IV. We describe a prediction model for CRC diagnosis using candidate biomarker peptides discovered by a one-step direct transfer technology (BLOTCHIP®-MS analysis). The high throughput technology has high reproducibility and is applicable for peptide quantification and differential analysis for biomarker discovery.
    Journal of Proteomics & Bioinformatics 03/2015; S5:1-8. DOI:10.4172/jpb.S5-005
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    ABSTRACT: Lysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion. K6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay. We identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359-378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt. Kgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.
    PLoS ONE 02/2015; 10(2):e0117775. DOI:10.1371/journal.pone.0117775 · 3.23 Impact Factor
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    ABSTRACT: To date, numerous studies have searched for candidate molecules or clinical examination methods as potential biomarkers for monitoring intractable diseases, such as carcinomas. Evidence accumulated over the past decade shows that many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. Although an analysis of the whole peptide (the 'peptidome') using mass spectrometry is thought to be one of the most powerful and promising experimental approaches, it has failed to identify biomarkers in the clinical blood samples, presumably due to the methodological limitations. In general, commonly used techniques for proteomic analysis of blood require the removal of large amounts of serum/plasma proteins prior to mass spectrometry analysis, and this step seems to have resulted in the overlooking of important biomarkers during the analytical process. Here, we provide a brief overview of a new quantitative peptidomic analysis by a one-step direct transfer technology without depletion of major blood proteins. Using this technology, we herein report experimental data on serum peptidomic analysis for patients with pregnancy-induced hypertension as a clinical model. In addition, we refer to the potential utility of this approach for the monitoring of pathophysiological status in female reproductive system disorders in general.
    Journal of Obstetrics and Gynaecology Research 09/2013; 39(10). DOI:10.1111/jog.12140 · 0.93 Impact Factor
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    ABSTRACT: We have recently developed a new target plate (BLOTCHIP®) for MALDI-MS. An advantage of this procedure is that it does not require the lowering of protein concentrations in test samples prior to analysis. Accordingly, this new technology enables the detection of peptides present in blood samples, including those that would otherwise be adsorbed to abundant blood proteins and would thus escape detection. Using this technology, we analyzed the peripheral blood of patients with pregnancy-induced hypertension (PIH; the most common serious complication of pregnancy) to test a potential utility of the technology for monitoring of the pathophysiological status. In the present study, we found 23 characteristic peptides for PIH in the blood serum of pregnant women. Offline LC-MALDI MS/MS identified 7 of the 23 peptides as fragments derived from kininogen-1 (three peptides), fibrinogen-α, complement component C4-A/B, α-2-HS-glycoprotein and inter-α-trypsin inhibitor heavy chain H4. 2-D scatter plots with combinations of the peptides found in the present study can be grouped for pregnant women with/without PIH, which would be satisfactory reflected for their status. Additionally, the levels of most of these peptides found were significantly decreased by albumin/IgG depletion prior to BLOTCHIP® analysis in accordance with conventional proteomics procedures. These results indicated that BLOTCHIP® analysis can be applied for discovery study of PIH biomarker candidates.
    Proteomics 07/2011; 11(13):2727-37. DOI:10.1002/pmic.201000753 · 3.97 Impact Factor
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    ABSTRACT: Human serum contains thousands of proteolytically derived low-molecular-weight peptide fragments (serum peptidome). The concept of utilizing the serum peptidome for cancer diagnosis has been developed. A pathological serum peptidome appears when the homeostatic balance between proteases and protease inhibitors is disrupted. We hypothesize if analyses of the serum peptidome are of diagnostic value as information on which molecules are disrupted, and the pathological course it will take in unknown pathological conditions and disseminated intravascular coagulation (DIC). We analyzed the serum peptidome in 3 stages (early stage, pre-DIC and DIC stages) in one patient with POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes) syndrome, an intractable disease with unknown pathology, using a 1-dimensional gel electrophoresis/matrix-assisted laser desorption/ionization-mass spectrometry (1-DE/MS)-based rapid quantitative approach. A very large number of peptide fragments appeared in the DIC stage, compared to pre-DIC. In addition, we identified fragments of transthyretin (ALGISPFHEHAEVVFTANDSGPR, m/z 2451.18) and alpha1-antitrypsin (EDPQGDAAQKTDTSHHDQDHPTFN, m/z 2691.02) that significantly increased in the DIC stage, compared to those in the pre-DIC stage. Rapid analyses of the serum peptidome may lead to a diagnostic method that can predict on-going protease activated pathological conditions and help to decide on multilateral strategies including nutritional support and drug therapy.
    Medical Hypotheses 06/2009; 73(5):760-3. DOI:10.1016/j.mehy.2009.04.026 · 1.07 Impact Factor
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    ABSTRACT: We have developed a new target plate for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This target plate enables direct electric transfer of analytes from the 1-dimensional gel electrophoresis (1-DE) gel to the target plate in one step. Incorporated with a one-step direct transfer technique, this novel 1-DE/MALDI-MS (1-DE/MS) system eliminates staining, extracting, loading, and many other time-consuming intermediate processes, thereby greatly reducing analysis time while providing high throughput proteome analysis. Furthermore, in peptidome analysis, during the 1-DE step this system separates or removes the high molecular weight plasma proteins in blood and the various low molecular weight substances in tissue extracts, which interfere with mass spectrometry. This system can therefore be used for peptide profiling of any biological sample without special pretreatment. In view of these advantages, the 1-DE/MS system will greatly improve the usefulness of current peptidomic modalities in the discovery and validation of biomarker molecules in various body fluids and tissue extracts, permitting early detection, diagnosis, and treatment of diseases.
    Biochemical and Biophysical Research Communications 01/2009; 379(1):110-4. DOI:10.1016/j.bbrc.2008.12.016 · 2.28 Impact Factor