Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from
            <i>Mycobacterium avium</i>
            subsp.
            <i>paratuberculosis</i>
            Strains K-10 and 187

Radosevich, Thomas J.; Reinhardt, Timothy A.; Lippolis, John D.; Bannantine, John P.; Stabel, Judith R.

doi:10.1128/jb.01420-06

Cited by 40 publications

(30 citation statements)

References 44 publications

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“…It is noted that the R.S.D. was even smaller than the typically reported 20% in iTRAQ experiments (15,34,35). The similarity of these distribution curves demonstrated the high reproducibility and precision of our quantitation strategy on the four independently prepared membrane samples.…”

Section: Assessment Of Reproducibility and Compatibility With Itraq Lmentioning

confidence: 67%

A Multiplexed Quantitative Strategy for Membrane Proteomics

Han

Chien

Chen³

et al. 2008

Molecular & Cellular Proteomics

130

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Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ (isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (<8% error) and precision (<12% relative S.D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate ‫؍‬ 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed >2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease.

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Section: Assessment Of Reproducibility and Compatibility With Itraq Lmentioning

confidence: 67%

A Multiplexed Quantitative Strategy for Membrane Proteomics

Han

Chien

Chen³

et al. 2008

Molecular & Cellular Proteomics

130

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“…Whole-cell sonicated extracts of mycobacterial species and isolates were prepared for use as antigens in immunoassays as described previously (30). Membrane-enriched and cytosol-enriched fractionated lysates were prepared as described previously (24).…”

mentioning

confidence: 99%

Production and Characterization of Monoclonal Antibodies against a Major Membrane Protein ofMycobacterium aviumsubsp.paratuberculosis

Bannantine

Radosevich

Stabel

et al. 2007

Clin Vaccine Immunol

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“…paratuberculosis cellular proteomes containing both cytoplasmic and membrane-enriched protein fractions (35)(36)(37). A total of 130 proteins overlapped with the cytoplasmic protein fractions from M. avium subsp.…”

Section: Discussionmentioning

confidence: 99%

Novel Secreted Antigens of Mycobacterium paratuberculosis as Serodiagnostic Biomarkers for Johne's Disease in Cattle

Facciuolo¹,

Kelton

Mutharia

2013

Clin Vaccine Immunol

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Johne's disease is a chronic gastroenteritis of cattle caused by Mycobacterium avium subsp. paratuberculosis that afflicts 40% of dairy herds worldwide. M. avium subsp. paratuberculosis-infected cattle can remain asymptomatic for years while transmitting the pathogen via fecal contamination and milk. Current serodiagnosis with enzyme-linked immunosorbent assays (ELISAs) fails to detect asymptomatic M. avium subsp. paratuberculosis-infected cattle due to the use of poorly defined antigens and knowledge gaps in our understanding of M. avium subsp. paratuberculosis components eliciting pathogen-specific immune responses. We set out to (i) define a subset of proteins that contain putative antigenic targets and (ii) screen these antigen pools for immunogens relevant in detecting infection. To accomplish our first objective, we captured and resolved M. avium subsp. paratuberculosis-secreted proteins using a 2-step fractionation method and reverse-phase liquid chromatography to identify 162 unique proteins, of which 66 had not been previously observed in M. avium subsp. paratuberculosis culture filtrates. Subsequent screening of M. avium subsp. paratuberculosis-secreted proteins showed four antigens, of which one or more reacted on immunoblotting with individual serum samples from 35 M. avium subsp. paratuberculosis-infected cows. Moreover, these novel antigens reacted with sera from 6 low M. avium subsp. paratuberculosis shedders and 3 fecal-culture-positive cows labeled as ELISA seronegative. The specificity of these antigens was demonstrated using negative-control sera from uninfected calves (n ‫؍‬ 5) and uninfected cows (n ‫؍‬ 5), which did not react to any of these antigens in immunoblotting. As three of the four antigens are novel, their characterization and incorporation into an ELISA-based format will aid in detecting asymptomatic cattle in early or subclinical stages of disease. E nzyme-linked immunosorbent assays (ELISAs) are simple, rapid, and cost-effective tests that have been used for decades for determination of infection status. One of the major challenges in the development of an effective ELISA is the selection of antigens that are pathogen specific and permit sensitive detection. Antibodies against shared epitopes in closely related species can contribute to cross-reactivity (resulting in false-positive identification), and fluctuations in antibody titers and antibody compositions in chronic diseases hinder the development of sensitive tests. These factors have been problematic for the development of ELISAs for all mycobacterial diseases, including human tuberculosis (Mycobacterium tuberculosis), leprosy (Mycobacterium leprae), bovine tuberculosis (Mycobacterium bovis), and Johne's disease (Mycobacterium avium subsp. paratuberculosis).Johne's disease is a chronic gastroenteritis affecting ruminant animals worldwide, with national prevalence rates of 32% in Canada and 68% in the United States (1, 2). Calves are most susceptible and, once colonized, they remain asymptomatic for 2 to 5 years while shedd...

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Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187

Cited by 40 publications

References 44 publications

A Multiplexed Quantitative Strategy for Membrane Proteomics

A Multiplexed Quantitative Strategy for Membrane Proteomics

Production and Characterization of Monoclonal Antibodies against a Major Membrane Protein ofMycobacterium aviumsubsp.paratuberculosis

Novel Secreted Antigens of Mycobacterium paratuberculosis as Serodiagnostic Biomarkers for Johne's Disease in Cattle

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