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Affinity Purification Mass Spectrometry on the Orbitrap-Astral Mass Spectrometer Enables High-Throughput Protein-Protein Interaction Mapping.
- Serrano, Lia;
- Pelin, Adrian;
- Arrey, Tabiwang;
- Damoc, Nicolaie;
- Richards, Alicia;
- Zhou, Yuan;
- Lancaster, Noah;
- Peters-Clarke, Trenton;
- Pashkova, Anna;
- Jang, Gwendolyn;
- Eckhardt, Manon;
- Quarmby, Scott;
- Zeller, Martin;
- Hermanson, Daniel;
- Stewart, Hamish;
- Hock, Christian;
- Makarov, Alexander;
- Zabrouskov, Vlad;
- Krogan, Nevan;
- Coon, Joshua;
- Swaney, Danielle
Abstract
Classical proteomics experiments offer high-throughput protein quantification but lack direct evidence of the spatial organization of the proteome, including protein-protein interaction (PPIs) networks. While affinity purification mass spectrometry (AP-MS) is the method of choice for generating these networks, technological impediments have stymied the throughput of AP-MS sample collection and therefore constrained the rate and scale of experiments that can be performed. Here, we build on advances in mass spectrometry hardware that have rendered high-flow liquid chromatography separations a viable solution for faster throughput quantitative proteomics. We describe our methodology using the Orbitrap-Astral mass spectrometer with 7 min, high-flow separations to analyze 216 AP-MS samples in ∼29 h. We show that the ion-focusing advancements, rapid mass analysis, and sensitive ion detection facilitate narrow-bin data-independent acquisition on a chromatographically practical timescale. Further, we highlight several aspects of state-of-the-art confidence-scoring software that warrant reinvestigation given the analytical characteristics of the Orbitrap-Astral mass spectrometer through comparisons with an enrichment-based thresholding technique. With our data, we generated an interaction map between 998 human proteins and 59 viral proteins. These results hold promise in expediting the throughput of AP-MS experiments, enabling more high-powered PPI studies.
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