Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids
Research output: Contribution to journal › Journal article › Research › peer-review
Standard
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.
In: Nature Communications, Vol. 14, 3599, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - Hybrid-DIA
T2 - intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids
AU - Martínez-Val, Ana
AU - Fort, Kyle
AU - Koenig, Claire
AU - Van der Hoeven, Leander
AU - Franciosa, Giulia
AU - Moehring, Thomas
AU - Ishihama, Yasushi
AU - Chen, Yu ju
AU - Makarov, Alexander
AU - Xuan, Yue
AU - Olsen, Jesper V.
N1 - Publisher Copyright: © 2023, The Author(s).
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
U2 - 10.1038/s41467-023-39347-y
DO - 10.1038/s41467-023-39347-y
M3 - Journal article
C2 - 37328457
AN - SCOPUS:85162073632
VL - 14
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
M1 - 3599
ER -
ID: 360682205