SureQuant Targeted Mass Spectrometry Standards and Assay Panel for Quantitative Analysis of Phosphorylated Proteins from Multiple Signaling Pathways

Importance of the Topic

The dynamic regulation of protein phosphorylation underlies critical signaling cascades that control cell growth, metabolism, stress responses, and apoptosis. Alterations in phosphorylation patterns drive disease processes including cancer, making reliable quantitation of phosphoproteomes essential for both basic research and translational applications.

Objectives and Study Overview

This study aimed to develop and evaluate Thermo Scientific SureQuant targeted mass spectrometry standards and workflows for accurate quantitation of phosphopeptides across multiple signaling pathways. Two heavy-labeled standard panels were assessed: a System Suitability Standard for LC-MS performance evaluation and a Multipathway Phosphopeptide Standard covering key sites in EGFR/HER, RAS-MAPK, PI3K/AKT/mTOR, AMPK, apoptosis, and stress pathways. The workflow integrates standardized sample preparation, optimized enrichment, and SureQuant IS-triggered acquisition.

Methods and Used Instrumentation

Sample Preparation and Enrichment:

• Cell lines (MCF-7, HCT-116, A431, LNCaP, HepG2) treated with IGF-1 or EGF, lysed with EasyPep buffer and phosphatase inhibitors.
• Digestion using EasyPep Maxi kit, spiking of 1 pmol multiplex standard.
• Phosphopeptide enrichment by Hi-Select Fe-NTA magnetic agarose, compared with SMOAC and competitor Fe-NTA methods.

Chromatography and Mass Spectrometry:

• Trap: Acclaim PepMap 100 C18; Analytical: EASY-Spray C18 or PepMap RSLC C18 column.
• LC systems: UltiMate 3000 RSLCnano, EASY-nLC 1200.
• Mass spectrometers: Q Exactive HF Hybrid Quadrupole-Orbitrap, Orbitrap Exploris 480.
• Acquisition modes: Data-Dependent Acquisition (DDA), Parallel Reaction Monitoring (PRM), SureQuant IS-triggered targeted MS.

Data Analysis:

• Proteome Discoverer 2.2 with SEQUEST HT for DDA.
• Skyline for SureQuant survey run optimization and light/heavy ratio measurements.

Main Results and Discussion

The System Suitability Standard provided comprehensive QC metrics, confirming LC-MS stability and sensitivity. The Multipathway Standard enabled detection and quantitation of 131 phosphorylation sites across diverse pathways. Enrichment with Hi-Select Fe-NTA beads outperformed SMOAC and competitor methods in yield and reproducibility. SureQuant IS-triggered acquisition achieved four-fold greater sensitivity than PRM while maintaining high precision, allowing robust quantitation of low-abundance phosphopeptides. Spike-in prior to enrichment maximized detection of targeted sites. Survey runs optimized precursor charge states and fragment selection to trigger high-resolution MS2 on endogenous targets only when standards reached threshold intensity, improving cycle efficiency.

Benefits and Practical Applications

This turnkey workflow offers:

• Standardized performance assessment of LC-MS systems for phosphoproteomics.
• Highly sensitive, multiplexed quantitation of biologically relevant phosphorylation events.
• Applicability to basic research, drug discovery, biomarker validation, and QA/QC in regulated environments.

Future Trends and Possibilities

Emerging directions include:

• Expansion of targeted panels to cover wider signaling networks and post-translational modifications.
• Integration with single-cell and spatial phosphoproteomics.
• Automation of enrichment and sample prep workflows.
• Incorporation of AI-driven data analysis for real-time decision making.
• Clinical translation for dynamic biomarker monitoring and personalized medicine.

Conclusion

The combination of heavy-labeled standards, optimized enrichment, and SureQuant targeted acquisition delivers a robust, sensitive, and precise platform for quantitative phosphoproteomics. This approach enables comprehensive monitoring of signaling dynamics across multiple pathways, supporting diverse research and clinical applications.