Serrano LR, Pelin A, Arrey TN, Damoc NE, Richards AL, Zhou Y, Lancaster NM, Peters-Clarke TM, Pashkova A, Jang GM, Eckhardt M, Quarmby ST, Zeller M, Hermanson D, Stewart H, Hock C, Makarov A, Zabrouskov V, Krogan NJ, Coon JJ, Swaney DL

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 ...

Read More

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.

Date: Apr. 04, 2025

View in: Pubmed Google Scholar

257220