Complementary proteomic analysis of protein complexes.

Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 917:391-407. DOI: 10.1007/978-1-61779-992-1_22
Source: PubMed

ABSTRACT Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The interferon-inducible protein IFI16 has emerged as a critical antiviral factor and sensor of viral DNA. IFI16 binds nuclear viral DNA, triggering expression of antiviral cytokines in response to infection with herpesviruses. Despite the relevance of IFI16 for understanding nucleus-derived immune response, the knowledge of the mechanisms through which IFI16 exerts its antiviral functions, and, specifically, of its protein interactions in the context of viral infection remains limited. Here, we provide the first characterization of endogenous IFI16 interactions following infection with the widely-spread human pathogen herpes simplex virus 1 (HSV-1). By integrating proteomics and virology approaches, we identify and validate IFI16 interactions with both viral and host proteins, reflective of the interplay between HSV-1 immunosuppressive mechanisms and host antiviral responses. For example, early during infection, IFI16 is targeted by the viral E3 ubiquitin ligase ICP0, in conjunction with a change in IFI16 localization via recruitment to ICP0-containing nuclear puncta. We observe that ICP0 is necessary, but not sufficient, for the proteasomal-mediated degradation of IFI16 following infection. By measuring antiviral cytokine expression, we substantiate that ICP0 suppresses IFI16-dependent immune responses. Indeed, infection with an HSV-1 strain that has an inactive ICP0 provides an environment in which IFI16 is not inhibited and is required for the ability of host cells to elicit interferon response. We next define temporal IFI16 interactions in this active immune signaling environment, determining its deposition at nuclear viral replication compartments. We discover and validate interactions with the viral protein ICP8 and cellular ND10 nuclear body components, both of which are markers for sites where HSV-1 deposits its DNA. These interactions may offer the necessary spatial co-localization for IFI16 to bind to nuclear viral DNA during infection. Altogether, our results provide critical insights into both viral inhibition of IFI16 and interactions that can contribute to IFI16 antiviral functions. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Molecular &amp Cellular Proteomics 02/2015; DOI:10.1074/mcp.M114.047068 · 7.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Transformation in most bacteria is dependent on orthologues of Type 2 secretion and Type 4 pilus system proteins. In each system, pilin proteins (major and minor) are required to make the pilus structure and are essential to the process, although the precise roles of the minor pilins remain unclear. We have explored protein-protein interactions among the competence minor pilins of Bacillus subtilis through in vitro binding studies, immunopurification and mass spectrometry. We demonstrate that the minor pilins directly interact, and the minor pilin ComGG interacts with most of the known proteins required for transformation. We find that ComGG requires other ComG proteins for its stabilization and for processing by the pre-pilin peptidase. These observations, C-terminal mutations in ComGG that prevent processing and the inaccessibility of pre-ComGG to externally added protease suggest a model in which pre-ComGG must be associated with other minor pilins for processing to take place. We propose that ComGG does not become a transmembrane protein until after processing. These behaviours contrast with that of pre-ComGC, the major pilin, which is accessible to externally added protease and requires only the peptidase to be processed. The roles of the pilins and of the pilus in transformation are discussed.
    Molecular Microbiology 10/2013; 90(6). DOI:10.1111/mmi.12425 · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.
    Development 02/2014; 141(4):962-73. DOI:10.1242/dev.098327 · 6.27 Impact Factor

Full-text (2 Sources)

Available from
Jun 3, 2014