• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1713-1719
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• 2006

A method for the simple, rapid, specific, and accurate detection of Bacillus anthracis spores was developed by employing specific capture peptides conjugated with fluorescent quantum dots (QDs). It was possible to distinguish B. anthracis spores from the spores of B. thuringiensis and B. cereus using these peptide-QD conjugates by flow cytometric and confocal laser scanning microscopic analyses. For more convenient high-throughput detection of B. anthracis spores, spectrofluorometric analysis of spore-peptide-QD conjugates was performed. B. anthracis spores could be detected in less than 1 h using this method. In order to avoid any minor yet false-positive signal caused by the presence of B. thuringiensis spores, the B-Negative peptide, which can only bind to B. thuringiensis, conjugated with another type of QD that fluoresces at different wavelength was also developed. In the presence of mixed B. anthracis and B. thuringiensis spores, the BABA peptide conjugated with QD525 and the B-Negative peptide conjugated with QD585 were able to bind to the former and the latter, specifically and respectively, thus allowing the clear detection of B. anthracis spores against B. thuringiensis spores by using two QD-labeling systems. This capture peptide-conjugated QD system should be useful for the detection of B. anthracis spores.

• Mass Spectrometry Letters
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• v.10 no.4
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• pp.117-122
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• 2019

This study demonstrated the sensitivity of electron capture dissociation mass spectrometry (ECD-MS) to probe subtle conformational changes in gaseous melittin ions induced by the substitution of an amino acid. ECD-MS was performed for triply and quadruply-protonated melittin and its variants obtained by a single amino acid substitution, namely, D-Pro14, Pro14Ala, and Leu13Ala. Although native triply-protonted melittin showed only a few peptide backbone cleavage products, the D-Pro14 and Pro14Ala variants exhibited extensive backbone fragments, suggesting the occurrence of a significant structural or conformational change induced by a single amino acid substitution at Pro14. On the contrary, the substitution at Leu13, namely Leu13Ala (+3), did not cause significant changes in the ECD backbone fragmentation pattern. Thus, the sensitivity of ECD-MS is demonstrated to be good enough to probe the aforementioned conformational change in melittin.

• Bulletin of the Korean Chemical Society
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• v.26 no.5
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• pp.740-746
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• 2005

In this article, we report the tandem mass spectrometry investigations for the electron capture efficiencies of the protons belonging to the different locations (generations) in a poly(propylene imine) dendrimer with three layers of a repeat unit (named as the third generation dendrimer). The employed tandem mass spectrometry methods include SORI-CAD (sustained off-resonance irradiation collisional activation dissociation) and ECD(electron capture dissociation) mass spectrometry. We obtained SORI-CAD spectra for the dendrimer ions in the different charge states, ranging from 2+ to 4+. The analysis of fragmentation sites provides the information as to where the protons are distributed among various generations of the dendrimer. Based upon this, a new strategy to study the electron capture efficiencies of the protons is utilized to examine a new type of triplycharged ions by SORI-CAD, i.e., the 3+ ions generated from the charge reduction of the native 4+ ions by ECD: (M+4H)$^{4+}\;+\;e^-\;{\rightarrow}$ (M+4H)$^{3+\bullet}$ ${\rightarrow}\;({H^{\bullet}}_{ejected}$) + (M+3H)$^{3+}\;\rightarrow$ CAD. Interestingly, comparison of these four SORICAD spectra indicates that the proton distribution in the charge-reduced 3+ ions is very close to that in the native 4+ ions. It further suggests that in this synthetic polymer ($\sim$1.7 kDa) with an artificial architecture, the electron capture efficiencies of the protons are actually insensitive to where they are located in the molecule. This is somewhat contradictory to common expectations that the protons in the inner generations may not be well exposed to the incoming electron irradiation as much as the outer ones are, thus may be less efficient for electron capture. This finding may carry some implications for the case of medium sized peptide ions with similar masses, which are known to show no obvious site-specific fragmentations in ECD MS.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• Journal of Biosystems Engineering
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• v.37 no.6
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• pp.420-428
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• 2012

Purpose: Porcine proliferative enteropathy(PPE), caused by the obligate intracellular bacterium Lawsonia intracellularis, is a widely distributed disease throughout the world causing substantial economic loss. The bacterial pathogen invades the intestinal epithelial cells which causes hyperplasia of the infected cells and leads to the process of disease pathogenesis. For diagnosing PPE in a pig farm in earlier stage, a rapid diagnosing test equipment is needed for farmers. To test the equipment appropriately, we prepare the samples and antibodies for rapid detecting the Lawsonia intracellularis in pig feces. Methods : To prepare the PPE infected samples, we sampled PPE suspected pig feces in a pig farm. To manufacture a anti-Lawsonia intracellularis antibody for capturing the Lawsonia intracellularis, the rabbit-anti LsaA synthetic peptide polyclonal antibody was inoculated to rabbits. To select the couple of antibodies which is most well sandwiched with the bacteria, ELISA test was done with PPE infected ileum samples. Finally, to verify the PPE infected feces which would be used to test the rapid kit, PCR test was done on the sampled PPE suspected feces Results : The rabbit-anti LsaA synthetic peptide polyclonal antibody is developed, and is verified to capture the bacterial well through the fluorescence antibody test. Also, we found that the monoclonal antibody and the polyclonal antibody could be used as couples for sandwiching the bacteria. Finally, through the PCR test for samples of pig feces, we could prepare the 150 PPE positive samples and 50 PPE negative samples. Conclusions : The manufactured polyclonal antibody and the imported monoclonal antibody could be used to capture the bacteria using the sandwich techniques. Also, the prepared PPE infected negative and positive samples could be used to test the performance of the rapid kit to capture the bacterium Lawsonia intracellularis.