Real-time, High Density Monitoring of pTyr Signaling Targets in Human Tumors Using Heavy Peptide Triggered Targeted Quantitation

Importance of the Topic

Protein tyrosine phosphorylation is a critical regulatory mechanism in cell signaling and is closely linked to oncogenic kinase activity, yet it represents less than 1 percent of total phosphorylation events. High-density, absolute quantitation of phosphotyrosine (pTyr) sites in clinical samples can reveal tumor-specific signaling alterations, improve reproducibility, and guide therapeutic decisions.

Objectives and Overview of the Study

This study aimed to establish a universal internal standard­guided LC-MS acquisition workflow for sensitive, reproducible, and high-density quantitation of pTyr targets in human colorectal tumor specimens. Key goals included enrichment of low-abundance pTyr peptides, spiking of heavy isotopic triggers, and implementation of real-time instrument control to quantify 356 pTyr sites per analysis.

Methodology and Instrumentation

Human colorectal tumor samples underwent protein extraction, reduction, alkylation, and tryptic digestion. A mixture of 356 stable isotopically labeled pTyr peptides (1 pmol) was spiked into each sample. Endogenous and heavy peptides were enriched by multiplexed immunoprecipitation using PT66, PY100, and 4G10 antibodies, followed by Fe-NTA metal affinity chromatography. A two-step Thermo Scientific SureQuant workflow was applied: a survey run to define optimal precursor charge states, fragment ions, and intensity thresholds, followed by IS-triggered acquisition alternating between fast watch mode for detection of heavy triggers and high-sensitivity quantitative mode for endogenous pTyr peptides. Instrumentation included the Thermo Scientific EASY-nLC 1200 system coupled to Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid mass spectrometers. Data processing and quantitation were performed in Skyline software.

Main Results and Discussion

The SureQuant workflow achieved robust detection of 356 pTyr targets across colorectal tumors with high sensitivity, yielding 6 to 8 data points per peak and enabling absolute concentration measurements via single-point calibration to heavy standards. Reproducibility exceeded 80 percent detection of targets across all samples, compared to under 25 percent for traditional TMT-based discovery analyses. Tumor-specific phosphorylation signatures, such as EGFR Y1173 levels, were quantified reliably, demonstrating the method’s capacity to profile signaling networks at high density without compromising selectivity or duty cycle.

Benefits and Practical Applications of the Method

• Simultaneous quantitation of hundreds of pTyr sites in a single LC-MS run
• Absolute concentration determination via isotopic internal standards
• Enhanced reproducibility and sensitivity compared to data-dependent and multiplexed discovery approaches
• Deployment on commercially available Orbitrap platforms with native control software

Future Trends and Potential Applications

The internal standard-triggered approach can be extended to other post-translational modifications such as serine and threonine phosphorylation, and to custom panels for disease or pathway-specific proteins. Integration with automated sample preparation and real-time adaptive acquisition strategies will further improve throughput and clinical applicability.

Conclusion

The SureQuant internal standard-guided workflow delivers sensitive, reproducible, and high-density targeted quantitation of phosphotyrosine signaling networks in tumor specimens. This strategy bridges the gap between global discovery and focused targeted proteomics, offering a versatile platform for comprehensive PTM analysis.

Reference

1. Vasaikar S., Huang C., Wang X., Petyuk V. A., Savage S. R., Wen B., et al. Proteogenomic Analysis of Human Colon Cancer Reveals New Therapeutic Opportunities. Cell. 2019.
2. Eid S., Turk S., Volkamer A., Rippmann F., Fulle S. KinMap: a web-based tool for interactive navigation through human kinome data. BMC Bioinformatics. 2017.