Protocol for mapping differential protein-protein interaction networks using affinity purification-mass spectrometry

Prashant Kaushal; Manisha R Ummadi; Gwendolyn M Jang; Yennifer Delgado; Sara K Makanani; Kareem Alba; Declan M Winters; Sophie F Blanc; Jiewei Xu; Benjamin Polacco; Yuan Zhou; Erica Stevenson; Manon Eckhardt; Lorena Zuliani-Alvarez; Robyn Kaake; Danielle L Swaney; Nevan J Krogan; Mehdi Bouhaddou

doi:10.1016/j.xpro.2024.103286

Protocol for mapping differential protein-protein interaction networks using affinity purification-mass spectrometry

STAR Protoc. 2024 Dec 20;5(4):103286. doi: 10.1016/j.xpro.2024.103286. Epub 2024 Nov 2.

Authors

Prashant Kaushal¹, Manisha R Ummadi², Gwendolyn M Jang², Yennifer Delgado³, Sara K Makanani³, Kareem Alba³, Declan M Winters³, Sophie F Blanc³, Jiewei Xu², Benjamin Polacco², Yuan Zhou², Erica Stevenson², Manon Eckhardt², Lorena Zuliani-Alvarez², Robyn Kaake⁴, Danielle L Swaney⁵, Nevan J Krogan⁶, Mehdi Bouhaddou⁷

Affiliations

¹ Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA; Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA, USA; Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA. Electronic address: [email protected].
² Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA; QBI Coronavirus Research Group (QCRG), University of California, San Francisco, San Francisco, CA, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA; Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA, USA.
³ Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA; Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA, USA; Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA.
⁴ Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA; QBI Coronavirus Research Group (QCRG), University of California, San Francisco, San Francisco, CA, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA; Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA, USA. Electronic address: [email protected].
⁵ Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA; QBI Coronavirus Research Group (QCRG), University of California, San Francisco, San Francisco, CA, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA; Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA, USA. Electronic address: [email protected].
⁶ Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA; QBI Coronavirus Research Group (QCRG), University of California, San Francisco, San Francisco, CA, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA; Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA, USA; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. Electronic address: [email protected].
⁷ Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA; Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA, USA; Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA. Electronic address: [email protected].

Abstract

Proteins congregate into complexes to perform diverse cellular functions. Protein complexes are remodeled by protein-coding mutations or cellular signaling changes, driving phenotypic outcomes in health and disease. We present an affinity purification-mass spectrometry (AP-MS) proteomics protocol to express affinity-tagged "bait" proteins in mammalian cells, identify and quantify purified protein interactors, and visualize differential protein-protein interaction networks between pairwise conditions. Our protocol possesses general applicability to various cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al.¹.

Keywords: Mass Spectrometry; Molecular Biology; Protein Biochemistry; Protein expression and purification; Proteomics; Systems biology.

MeSH terms

Chromatography, Affinity* / methods
HEK293 Cells
Humans
Mass Spectrometry* / methods
Protein Interaction Mapping* / methods
Protein Interaction Maps* / physiology
Proteins / metabolism
Proteomics* / methods

Substances

Proteins