Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids
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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.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 3599 |
| Tidsskrift | Nature Communications |
| Vol/bind | 14 |
| Antal sider | 18 |
| ISSN | 2041-1723 |
| DOI | |
| Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:
The project was part of a research collaboration 'cSHARP' between Thermo Fisher Scientific, University of Copenhagen, Kyoto University and Academia Sinica. Work at The Novo Nordisk Foundation Center for Protein Research (CPR) is funded in part by a generous donation from the Novo Nordisk Foundation (NNF14CC0001). This work has also been funded as part of EPIC-XS project under the grant agreement no. 823839 funded by the Horizon 2020 programme of the European Union and supported by the European Research Council through ERC-Synergy grant 810057-HighResCells. C.K. is supported by the Marie Skłodowska Curie European Training Network “PUSHH” (grant number No. 861389). We thank Aaron Gajadhar and Bhavin Patel from Thermo Fisher Scientific for providing us early access to the SureQuant™ Multipathway Phosphopeptide Standard kit.
Funding Information:
The project was part of a research collaboration 'cSHARP' between Thermo Fisher Scientific, University of Copenhagen, Kyoto University and Academia Sinica. Work at The Novo Nordisk Foundation Center for Protein Research (CPR) is funded in part by a generous donation from the Novo Nordisk Foundation (NNF14CC0001). This work has also been funded as part of EPIC-XS project under the grant agreement no. 823839 funded by the Horizon 2020 programme of the European Union and supported by the European Research Council through ERC-Synergy grant 810057-HighResCells. C.K. is supported by the Marie Skłodowska Curie European Training Network “PUSHH” (grant number No. 861389). We thank Aaron Gajadhar and Bhavin Patel from Thermo Fisher Scientific for providing us early access to the SureQuant™ Multipathway Phosphopeptide Standard kit.
Publisher Copyright:
© 2023, The Author(s).
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