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

• Published in: Nature communications Vol. 14; no. 1; p. 3599
• Main Authors: Martínez-Val, Ana, Fort, Kyle, Koenig, Claire, Van der Hoeven, Leander, Franciosa, Giulia, Moehring, Thomas, Ishihama, Yasushi, Chen, Yu-ju, Makarov, Alexander, Xuan, Yue, Olsen, Jesper V.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 16-06-2023; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 631/154/1435; 631/1647/2067; 631/1647/296; 631/80/458/1733; 64; 82/47; 82/58; 96; Application programming interface; Cancer; Cell culture; Chemotherapy; Data acquisition; Drug screening; Humanities and Social Sciences; Image reconstruction; Ions; Mass spectrometry; Mass spectroscopy; multidisciplinary; Phosphorylation; Proteomics; Science; Science (multidisciplinary); Scientific imaging; Sensitivity; Signal transduction; Signaling; Spheroids; Stable isotopes; Target acquisition
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-39347-y

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.
Standard mass spectrometry analyses often miss key targets required for phospho-signalling reconstruction. Here, authors present an intelligent data acquisition strategy that combines discovery and targeted analysis in one run and apply it to maximize the information from single spheroids drug screenings.