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Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.
Kaushal, Prashant; Ummadi, Manisha R; Jang, Gwendolyn M; Delgado, Yennifer; Makanani, Sara K; Blanc, Sophie F; Winters, Decan M; Xu, Jiewei; Polacco, Benjamin; Zhou, Yuan; Stevenson, Erica; Eckhardt, Manon; Zuliani-Alvarez, Lorena; Kaake, Robyn; Swaney, Danielle L; Krogan, Nevan; Bouhaddou, Mehdi.
Afiliação
  • Kaushal P; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
  • Ummadi MR; Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA, USA.
  • Jang GM; Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA.
  • Delgado Y; Technical Contact.
  • Makanani SK; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA.
  • Blanc SF; QBI Coronavirus Research Group (QCRG), University of California, San Francisco, San Francisco, CA, USA.
  • Winters DM; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
  • Xu J; Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA, USA.
  • Polacco B; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA.
  • Zhou Y; QBI Coronavirus Research Group (QCRG), University of California, San Francisco, San Francisco, CA, USA.
  • Stevenson E; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
  • Eckhardt M; Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA, USA.
  • Zuliani-Alvarez L; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
  • Kaake R; Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA, USA.
  • Swaney DL; Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA.
  • Krogan N; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
  • Bouhaddou M; Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA, USA.
ArXiv ; 2024 May 15.
Article em En | MEDLINE | ID: mdl-38800652
ABSTRACT
Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein-coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification-mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein-protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity-tagged "bait" proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity-tagged "bait" gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross-run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 20231.
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Texto completo: 1 Base de dados: MEDLINE Idioma: En Ano de publicação: 2024 Tipo de documento: Article
Texto completo
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XML
PubMed Links
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Texto completo: 1 Base de dados: MEDLINE Idioma: En Ano de publicação: 2024 Tipo de documento: Article