Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry
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| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Preprint |
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2024
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| _version_ | 1866909205332492288 |
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| author | 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 |
| author_facet | 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 |
| contents | 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. |
| format | Preprint |
| id |
arxiv_https___arxiv_org_abs_2405_09699 |
| institution | arXiv |
| publishDate | 2024 |
| record_format | arxiv |
| spellingShingle | 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 Quantitative Methods Molecular Networks 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. |
| title | Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry |
| topic | Quantitative Methods Molecular Networks |
| url | https://arxiv.org/abs/2405.09699 |