A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms |
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Authors: | West Rolieria Whitmon Jennifer Williamson Yulanda M Moura Hercules Nelson Marguerite Melnick Nikkol Tondella Maria Lucia C Schieltz David Rees Jon Woolfitt Adrian R Barr John R Ades Edwin W Carlone George M Sampson Jacquelyn S |
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Affiliation: | a Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USAb Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Chamblee, Georgia 30341, USA |
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Abstract: | Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins. |
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Keywords: | Bordetella pertussis Immunoprecipitation Immunoproteomics |
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