Affinity Purification Mass Spectrometry on the Orbitrap-Astral Mass Spectrometer Enables High-Throughput Protein-Protein Interaction Mapping.

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.