Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Wilmarth, Phillip A.; Riviere, Michael A.; David, Larry L.

doi:10.1007/s12177-009-9042-6

Cited by 96 publications

(108 citation statements)

References 36 publications

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“…Sequest (version 28, revision 12; Thermo Scientific) was used to search MS2 spectra against a May 2014 version of the Sprot human FASTA protein database, with added sequences for E. coli BirA* and HIV-1 strain NL4-3, concatenated sequence-reversed entries to estimate error thresholds, and 179 common contaminant sequences and their reversed forms. The database processing was performed with Python scripts that have been described previously (65). Searches for all samples were performed with trypsin enzyme specificity.…”

Section: Methodsmentioning

confidence: 99%

“…A variable modification of ϩ16 Da on methionine residues was also allowed, with a maximum of 3 modifications per peptide. A linear discriminant transformation was used to improve the identification sensitivity from the SEQUEST analysis (65,66). SEQUEST scores were combined into linear discriminant function scores, and discriminant score histograms were created separately for each peptide charge state (1ϩ, 2ϩ, and 3ϩ).…”

Section: Methodsmentioning

confidence: 99%

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Analysis of HIV-1 Gag Protein Interactions via Biotin Ligase Tagging

Ritchie

Cylinder

Platt

et al. 2015

J Virol

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We have examined the interactions of wild-type (WT) and matrix protein-deleted (⌬MA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ⌬MA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ⌬MA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Analysis of HIV-1 Gag Protein Interactions via Biotin Ligase Tagging

Ritchie

Cylinder

Platt

et al. 2015

J Virol

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“…The database processing was performed with python scripts available at http://www.proteomicanalysisworkbench.com. Comet searches for all samples were performed with trypsin enzyme specificity (28,29). Peptide-to-protein mapping and protein filtering were performed using PAW_results_7.py (version 7.0).…”

Section: Analysis Of Proteinmentioning

confidence: 99%

Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

Geszvain

Smesrud

Tebo

2016

Appl Environ Microbiol

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The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ⌬fleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. IMPORTANCEThe identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase-and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis in Pseudomonas aeruginosa. Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation.

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“…Peptide-to-protein mapping and protein filtering were performed using PAW_results_6.py (version 6.1). The in-house Python scripts have been described previously (27). The overall probability of protein identification was calculated by dividing the total number of MS/MS spectra matching the protein by the total number of matching MS/MS spectra unique to the protein.…”

mentioning

confidence: 99%

Interaction of Mycobacterium leprae with Human Airway Epithelial Cells: Adherence, Entry, Survival, and Identification of Potential Adhesins by Surface Proteome Analysis

et al. 2013

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f This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

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Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Cited by 96 publications

References 36 publications

Analysis of HIV-1 Gag Protein Interactions via Biotin Ligase Tagging

Analysis of HIV-1 Gag Protein Interactions via Biotin Ligase Tagging

Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

Interaction of Mycobacterium leprae with Human Airway Epithelial Cells: Adherence, Entry, Survival, and Identification of Potential Adhesins by Surface Proteome Analysis

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