Simultaneous targeted and discovery phosphoproteomics of cell signaling pathways using novel hybrid-DIA acquisition strategy

Importance of the Topic

Modern cell signaling research demands analytical approaches capable of both deep discovery of global phosphorylation patterns and precise quantification of predefined regulatory sites. Combining targeted and untargeted proteomics workflows in a single experiment reduces sample consumption and instrument time while ensuring robust measurement of both known and novel phosphorylation events.

Study Objectives and Overview

This study introduces and benchmarks a hybrid data-independent acquisition (Hybrid DIA) strategy that interleaves conventional wide-window DIA with accurately triggered scans of user-defined heavy and endogenous peptide pairs. The approach was applied to HeLa cell lysates stimulated with epidermal growth factor (EGF) under kinase-inhibitor perturbations to:

• Quantify 131 biologically relevant phosphopeptide targets over two time points.
• Assess sensitivity against a purely targeted method (SureQuant).
• Perform a comprehensive library-free phosphoproteome analysis.

Methodology and Instrumentation

Enzymatic digestion of 3–4 million HeLa cells was performed using LysC and trypsin, followed by TiIMAC-based phosphopeptide enrichment. Hybrid DIA runs were acquired on a high-resolution orbitrap mass spectrometer via an API that triggers MSx scans for selected m/z and fragment ions. Acquisition parameters included:

• Interleaved DIA windows covering MS1 and full MS2 scans.
• Targeted MSx scans corrected by injection time normalization.
• Timed retention-time windows for heavy/light pairs.

Raw data were converted to mzML by MSx_extractor.py (pymsfilereader, ProteoWizard) and analyzed in Skyline for fragment extraction and heavy/light ratio calculation. Downstream processing employed a custom shinyApp (R) for normalization and RoKAI App v2.1.3 for kinase activity inference.

Main Results and Discussion

Hybrid DIA demonstrated improved sensitivity and lower limits of detection compared to SureQuant, particularly for low-abundance phosphopeptides. Key findings include:

• Consistent quantification of 131 targeted sites across 0, 10, and 90 min post-EGF stimulation with mean heavy/light ratios and normalized AUC.
• Identification of 5 055 phosphosites (localization probability > 0.75) in the full phosphoproteome, of which 3 611 were quantified in ≥ 75% of replicates.
• Detection of 1 214 significantly regulated phosphosites (ANOVA FDR < 0.05%) and 224 sites activated after 10 min EGF treatment.
• Inference of differential kinase activities, highlighting modulation of EGFR, MEK, PI3K, AKT, and downstream MAPKs under various inhibitor treatments.

Benefits and Practical Applications

Hybrid DIA unifies targeted and discovery proteomics, offering:

• Simultaneous measurement of predefined biomarkers and global signaling changes.
• Reduced sample input and acquisition time compared to separate targeted and DIA runs.
• Enhanced throughput for temporal and perturbation studies in QA/QC, drug discovery, and basic research.

Future Trends and Opportunities

Advances may include real-time acquisition decisions guided by machine learning, expanded peptide inclusion lists for multiplexed quantification, integration with single-cell and imaging proteomics, and fusion with other omics layers. Further development of streamlined software pipelines will improve accessibility for non-specialized laboratories.

Conclusion

Hybrid DIA effectively bridges the gap between targeted and discovery workflows, delivering high sensitivity for regulatory sites while maintaining broad coverage of the phosphoproteome. This approach accelerates comprehensive signaling studies and supports diverse applications in analytical chemistry and proteomics.