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Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids
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 (hy...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10276052/ https://www.ncbi.nlm.nih.gov/pubmed/37328457 http://dx.doi.org/10.1038/s41467-023-39347-y |
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author | 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. |
author_facet | 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. |
author_sort | Martínez-Val, Ana |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-10276052 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-102760522023-06-18 Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids 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. Nat Commun Article 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. Nature Publishing Group UK 2023-06-16 /pmc/articles/PMC10276052/ /pubmed/37328457 http://dx.doi.org/10.1038/s41467-023-39347-y Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article 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. Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids |
title | Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids |
title_full | Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids |
title_fullStr | Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids |
title_full_unstemmed | Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids |
title_short | Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids |
title_sort | hybrid-dia: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10276052/ https://www.ncbi.nlm.nih.gov/pubmed/37328457 http://dx.doi.org/10.1038/s41467-023-39347-y |
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