Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
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
_version_ 1785059995163820032
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
work_keys_str_mv AT martinezvalana hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT fortkyle hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT koenigclaire hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT vanderhoevenleander hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT franciosagiulia hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT moehringthomas hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT ishihamayasushi hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT chenyuju hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT makarovalexander hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT xuanyue hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids
AT olsenjesperv hybriddiaintelligentdataacquisitionintegratestargetedanddiscoveryproteomicstoanalyzephosphosignalinginsinglespheroids