hybrid-DIA

hybrid-DIA integrates targeted and discovery proteomics using a hybrid data-independent acquisition (DIA) strategy to enable quantitative phosphoproteome profiling and targeted analysis of predefined phosphopeptide standards.

Key Features:

• Integration of Targeted and Discovery Proteomics: Uses an Application Programming Interface (API) to dynamically intercalate data-independent acquisition (DIA) scans with multiplexed tandem mass spectrometry (MSx) scans, combining targeted acquisition of predefined phosphopeptide standards with global phosphoproteome profiling.
• Dynamic Triggering Mechanism: Implements an accurate triggering mechanism for MSx scans to precisely target specific phosphopeptides and improve quantitative accuracy and sensitivity.
• Benchmarking Against State-of-the-Art Methods: Benchmarked against SureQuant using heavy stable isotope-labeled phosphopeptide standards spanning seven major signaling pathways, demonstrating comparable quantitative accuracy and sensitivity while providing comprehensive phosphoproteome coverage.
• Application in Biomedical Research: Applied to assess effects of chemotherapeutic agents on single colon carcinoma multicellular spheroids and to evaluate differences in phospho-signaling between 2D and 3D cell cultures.

Scientific Applications:

• Signaling pathway reconstruction: Enables reconstruction of signaling pathways through quantitative phosphoproteomic measurements using both targeted standards and global DIA data.
• Limited-sample phosphoproteomics: Facilitates analysis of small sample amounts, including single multicellular spheroids, via targeted MSx acquisition.
• Comparative culture and cancer studies: Supports comparative studies of phospho-signaling between 2D and 3D cell culture models and pharmacological perturbations such as chemotherapeutic agents in cancer research.
• Quantitative targeted and global profiling: Combines targeted quantification of predefined phosphopeptide standards with global phosphoproteome profiling for sensitive and comprehensive phosphorylation analysis.

Methodology:

An API dynamically intercalates DIA scans with multiplexed MSx scans and implements an MSx triggering mechanism; heavy stable isotope-labeled phosphopeptide standards are used for targeted acquisition and quantitation.