Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids

Ana Martínez-Val, Kyle Fort, Claire Koenig, Leander Hoeven, Giulia Franciosa, Thomas Moehring, Yasushi Ishihama, Yu-ju Chen, Alexander Makarov, Yue Xuan () and Jesper V. Olsen ()
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Ana Martínez-Val: University of Copenhagen
Kyle Fort: Thermo Fisher Scientific
Claire Koenig: University of Copenhagen
Leander Hoeven: University of Copenhagen
Giulia Franciosa: University of Copenhagen
Thomas Moehring: Thermo Fisher Scientific
Yasushi Ishihama: Kyoto University
Yu-ju Chen: Academia Sinica
Alexander Makarov: Thermo Fisher Scientific
Yue Xuan: Thermo Fisher Scientific
Jesper V. Olsen: University of Copenhagen

Nature Communications, 2023, vol. 14, issue 1, 1-18

Abstract: Abstract Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

Date: 2023
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DOI: 10.1038/s41467-023-39347-y

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