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Nishi-Tokyo-shi, Japan

Kishimoto T.,Molecuence Corporation | Kishimoto T.,Mitsubishi Group | Kishimoto T.,Tokyo Medical and Dental University | Kondo J.,Mitsubishi Group | And 2 more authors.
Proteomics | Year: 2011

Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Katano M.,St. Marianna University School of Medicine | Katano M.,Mitsubishi Group | Katano M.,Molecuence Corporation | Okamoto K.,St. Marianna University School of Medicine | And 7 more authors.
Clinical and Experimental Rheumatology | Year: 2011

Objectives: In our previous proteomic surveillance, we found that at least 11 proteins in neutrophils were increased more than 2.5-fold by the stimulation of GM-CSF. In this paper, focusing on one of the 11 proteins, SlOO calcium binding protein A8 (S100A8), we tried to elucidate the effect of SI00A8 and the cooperative effect of S100A8 and GM-CSF on production and secretion of cytokines of neutrophils. Methods: S100A8 in neutrophil was detected by western blotting, and concentrations of SI00A8 in synovial fluid (SF)from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were measured by EL1SA. Cytokine levels in the culture medium of neutrophils incubated with and without S100A8 were measured by an antibody array. IL-8 and IL-16 levels in the culture medium of neutrophils stimulated with S100A8, GM-CSF, and the combination of S100A8 and GM-CSF were measured by ELISA. The mRNA levels of IL-8 and IL-16 in the stimulated neutrophils were analysed by real-time PCR. Results: The western blotting analysis confirmed that S100A8 is up-regulated in neutrophil by the stimulation of GM-CSF. Furthermore, the ELISA analysis confirmed that S100A8 was significantly elevated in SF of patients with RA compared to SF of patients with OA. S100A8 induced mRNA expression and secretion of IL-8 and IL-16. S100A8 further enhanced production of IL-8 by GM-CSF but not that of IL-16. Conclusions: These data suggest that SI00A8 may be involved in the exacerbation of RA, and that S100A8 may be a therapeutic target of RA. © Copyright CUN1CAL AND EXPERIMENTAL RHEUMATOLOGY 2011.

Kobayashi T.,ZoeGene Corporation | Kobayashi T.,Molecuence Corporation | Kakui M.,ZoeGene Corporation | Shibui T.,ZoeGene Corporation | And 2 more authors.
Molecular Biotechnology | Year: 2011

Interleukin-6 (IL-6) plays a crucial role in malignant diseases, such as rheumatoid arthritis, Castleman disease, and multiple myeloma, and as such, is an attractive therapeutic target. Here, the authors isolated a novel IL-6 inhibitor peptide by in vitro selection using mRNA display. The authors first used a random-primed human cDNA library to isolate IL-6-binding peptides. After four rounds of selection, a 19-amino acid peptide named CA11 was selected and confirmed to specifically interact with IL-6. The authors then performed an alanine scan analysis of CA11 and determined the amino acid residues necessary to interact with IL-6. Next, the authors constructed a CA11-based partially randomized library and after ten more rounds of selection, isolated several groups of peptides. The most frequently occurring sequence, RA07, bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130. © 2010 Springer Science+Business Media, LLC.

Matsumura N.,Keio University | Matsumura N.,Hokkaido University | Tsuji T.,Keio University | Sumida T.,Keio University | And 7 more authors.
FASEB Journal | Year: 2010

Bcl-XL, an antiapoptotic member of the Bcl-2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16-mer peptides that interact with Bcl-XL from random and degenerate peptide libraries using mRNA display. The selected peptides have sequence similarity with the Bcl-2 family BH3 domains, and one of them has higher affinity (IC50=0.9 μM) than Bak BH3 (IC50=11.8 μM) for Bcl-XL in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl-XL, localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl-X L. These results demonstrate that this selected peptide specifically antagonizes the function of Bcl-XL and overcomes the effects of Bcl-XL in intact cells. We suggest that mRNA display is a powerful technique to identify peptide inhibitors with high affinity and specificity for disease-related proteins. © FASEB.

Honda K.,National Cancer Center Research Institute | Okusaka T.,National Cancer Center Hospital | Felix K.,University of Heidelberg | Nakamori S.,Osaka National Hospital | And 15 more authors.
PLoS ONE | Year: 2012

Background: Among the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection. Methods: We compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1) using a newly developed matrix-assisted laser desorption/ionization (oMALDI) QqTOF (quadrupole time-of-flight) mass spectrometry (MS) system. Results: We found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10-21, P = 4.35×10-14, and P = 1.83×10-24 (Mann-Whitney U-test); area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a prospective cohort [Cohort 4 (n = 833)] collected from 8 medical institutions in Japan and Germany. Conclusions: We have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer. © 2012 Honda et al.

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