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Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

ABSTRACT: Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging when analyzing tiny sample amounts. We present a novel 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 MS scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmarked hybrid-DIA against state-of-the-art targeted MS methods (i.e. SureQuant) using EGF-stimulated HeLa cells and found the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiled the global phosphoproteome. To demonstrate the robustness, sensitivity and potential of hybrid-DIA, we profiled chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluated the difference of cancer cells in 2D vs 3D culture. Altogether, we showed that hybrid-DIA is the way-to-go method in highly sensitive phospho-proteomics experiments.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ana Martinez-Val

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD038947 | Pride | 2023-05-18

REPOSITORIES: Pride

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			Action	DRS
	20220131_EXPL8_Evo3_AMV_20SPD_SQ_Phos_HeLa_CTRL_0-05pmol131pept-bef_01.raw	Raw
	20220131_EXPL8_Evo3_AMV_20SPD_SQ_Phos_HeLa_CTRL_0-05pmol131pept-bef_02.raw	Raw
	20220131_EXPL8_Evo3_AMV_20SPD_SQ_Phos_HeLa_EGF10min_0-05pmol131pept-bef_01.raw	Raw
	20220131_EXPL8_Evo3_AMV_20SPD_SQ_Phos_HeLa_EGF10min_0-05pmol131pept-bef_02.raw	Raw
	20220131_EXPL8_Evo3_AMV_20SPD_SQ_Phos_HeLa_EGF90min_0-05pmol131pept-bef_01.raw	Raw

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Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

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