title: A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms.
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.
authors.affiliation: Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
authors.forename: Rolieria
authors.initials: R
authors.lastname: West
authors.forename: Jennifer
authors.initials: J
authors.lastname: Whitmon
authors.forename: Yulanda M
authors.initials: YM
authors.lastname: Williamson
authors.forename: Hercules
authors.initials: H
authors.lastname: Moura
authors.forename: Marguerite
authors.initials: M
authors.lastname: Nelson
authors.forename: Nikkol
authors.initials: N
authors.lastname: Melnick
authors.forename: Maria Lucia C
authors.initials: ML
authors.lastname: Tondella
authors.forename: David
authors.initials: D
authors.lastname: Schieltz
authors.forename: Jon
authors.initials: J
authors.lastname: Rees
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Woolfitt
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
authors.forename: Edwin W
authors.initials: EW
authors.lastname: Ades
authors.forename: George M
authors.initials: GM
authors.lastname: Carlone
authors.forename: Jacquelyn S
authors.initials: JS
authors.lastname: Sampson
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification*; D001886:Bordetella pertussis / Q000276:immunology*; D047468:Immunoprecipitation / Q000379:methods; D036103:Nanotechnology; D040901:Proteomics / Q000379:methods; D011817:Rabbits; D053719:Tandem Mass Spectrometry
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins