Advanced Phosphoproteomics Depth and Quantitative Performance Using an Integrated noFAIMS-FAIMS Approach

Area of Science:

• Proteomics
• Mass Spectrometry
• Cellular Signaling

Background:

• Protein phosphorylation is crucial for cellular regulation and implicated in diseases like cancer.
• Detecting low-abundance phosphopeptides via mass spectrometry remains challenging.
• High-field asymmetric waveform ion mobility spectrometry (FAIMS) improves phosphopeptide detection but has limitations.
• Combining FAIMS with conventional non-FAIMS analysis offers complementary data.

Purpose of the Study:

• To evaluate the efficacy of integrating non-FAIMS and FAIMS approaches for enhanced phosphoproteomic analysis.
• To determine if combining these methods increases the depth and breadth of phosphopeptide identification.
• To assess the impact of integrated analysis on the coverage of key cellular signaling pathways.

Main Methods:

• Phosphopeptide samples were analyzed using UHPLC coupled to an Orbitrap Eclipse Tribrid mass spectrometer.
• Ionization efficiencies were assessed using parallel reaction monitoring (PRM) with synthetic phosphopeptides under various FAIMS conditions.
• The integrated approach was validated using data-dependent acquisition (DDA) on HEK293 and HeLa cell phosphopeptide samples.

Main Results:

• The integrated non-FAIMS-FAIMS approach increased phosphopeptide identifications by 14.9%-46.5% compared to individual methods.
• The integrated strategy demonstrated high reproducibility across replicates.
• Key signaling pathways (e.g., EGF/EGFR, VEGFA-VEGFR2, PI3K-AKT) showed expanded coverage by capturing unique phosphoproteins.

Conclusions:

• Integrating non-FAIMS and FAIMS significantly enhances phosphoproteomic depth and quantification.
• This strategy effectively leverages complementary data from both methods.
• The integrated approach provides a practical and efficient means for deeper insights into cellular signaling.