Systematic analysis of the use of amphipathic polymers for studies of outer membrane proteins using mass spectrometry

ABSTRACT Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4+ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.

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Rapid typing ofMoraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry

PROTEOMICS ◽

10.1002/pmic.200500086 ◽

2006 ◽

Vol 6 (1) ◽

pp. 172-180 ◽

Cited By ~ 21

Author(s):

André Schaller ◽

Rolf Troller ◽

Daniel Molina ◽

Sabina Gallati ◽

Christoph Aebi ◽

...

Keyword(s):

Mass Spectrometry ◽

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins

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Identification of Immunodominant Outer Membrane Proteins of Fusobacterium necrophorum from Severe Ovine Footrot By MALDI-TOF Mass Spectrometry

Current Microbiology ◽

10.1007/s00284-021-02383-2 ◽

2021 ◽

Vol 78 (4) ◽

pp. 1298-1304

Author(s):

S. Farooq ◽

S. A. Wani ◽

S. Qureshi ◽

M. A. Bhat ◽

Z. A. Kashoo ◽

...

Keyword(s):

Mass Spectrometry ◽

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Fusobacterium Necrophorum ◽

Effective Control ◽

Foot Rot ◽

Sequence Coverage ◽

Maldi Tof ◽

Flight Mass Spectrometry

AbstractThe aim of this study was to identify the immunodominant outer membrane proteins (OMPs) of Fusobacterium necrophorum from sheep affected with severe foot-rot. The OMP profile of ovine strains of F. necrophorum has not been well studied. We analyzed the OMP profile of the most frequent lktA variant JKS-F3 of F. necrophorum associated with severe ovine foot-rot with lesion score 4 in order to identify its major immunodominant OMPs. Electrophoretic separations of extracted OMPs showed a number of spots in two-dimensional electrophoretic gels. Two immunoreactive proteins of size around 43 kDa were identified through western blotting using hyperimmune sera raised in rabbits. These two immunogenic OMPs were analyzed by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF/MS) which revealed that these two OMPs of lktA variant JKS-F3 of F. necrophorum showed 46 and 42 percent protein sequence coverage and scores of 125 and 114, respectively, with the reported 43 kDa outer membrane protein of F. necrophorum strain H05, a putative porin having properties similar to pore-forming proteins of anaerobic Gram-negative bacteria. These identified immunogenic OMPs will contribute to our understanding of the pathogenic role played by this organism in ovine foot-rot and could be exploited to devise an effective control strategy through development of an OMP-based recombinant vaccine to mitigate foot-rot in sheep and goats.

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Short-Chain Fatty Acids Modulate Metabolic Pathways and Membrane Lipids in Prevotella bryantii B14

Proteomes ◽

10.3390/proteomes8040028 ◽

2020 ◽

Vol 8 (4) ◽

pp. 28

Author(s):

Andrej Trautmann ◽

Lena Schleicher ◽

Simon Deusch ◽

Jochem Gätgens ◽

Julia Steuber ◽

...

Keyword(s):

Mass Spectrometry ◽

Fatty Acids ◽

Amino Acid ◽

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Short Chain Fatty Acids ◽

Isovaleric Acid ◽

Short Chain ◽

Prevotella Bryantii

Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B14 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid.

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