Evaluation of Parallel Reaction Monitoring assays at discovery scale on a new hybrid nominal mass instrument for phosphoproteomics studies

Importance of the Topic
Understanding protein phosphorylation at large scale is fundamental for decoding cellular signaling pathways and disease mechanisms. High‐throughput, quantitative phosphoproteomics enables researchers to monitor dynamic changes in phosphorylation and supports the discovery of therapeutic targets and biomarkers.

Study Aims and Overview
This work evaluates discovery‐scale parallel reaction monitoring (PRM) workflows on a new hybrid nominal mass spectrometer, comparing traditional targeted MS2 (tMS2) with synchronous precursor selection MS3 (tMS3) acquisition. The goal is to establish robust assays for absolute quantitation of over 300 phosphopeptides using stable isotope‐labeled (SIL) standards across cancer cell line samples.

Methodology and Instrumentation
A spectral library of 335 synthetic SIL phosphopeptides was built on a high‐resolution accurate mass (HRAM) platform. PRM Conductor, a Skyline‐based plugin, generated scheduled PRM and tMS3 methods by refining transitions according to peak shape, signal‐to‐noise, and ion ratios. Samples were separated on a Thermo Scientific Easy‐Spray PepMap Neo C18 column (75 µm × 150 mm) using a 30-min gradient on a Vanquish Neo UHPLC system. Data acquisition employed the Thermo Scientific Stellar hybrid mass spectrometer, using HCD for tMS2 and synchronous precursor selection for tMS3. Endogenous peptide enrichment from five cancer cell lines was performed via immobilized metal affinity chromatography (IMAC).

Key Results and Discussion
The assays demonstrated excellent linearity from 18 amol to 40 fmol on column, with limits of quantitation in the low attomole range. PRM Conductor refinement improved selectivity by removing noisy transitions, enhancing signal quality. Comparison of tMS2 and tMS3 revealed better specificity and lower limits of quantitation with tMS3 for certain peptides. Reproducibility was high at both 494 amol and 13.3 fmol levels. In the five-cell-line mixture, the number of detected endogenous phosphopeptides scaled with input amount: 113 at 25 µg up to 193 at 500 µg, indicating at least four-fold sensitivity gain over existing triple quadrupole methods.

Benefits and Practical Applications

• High‐throughput quantitation of >300 phosphopeptides in a 30-min run
• Improved sensitivity in the attomole range, suitable for low‐abundance targets
• Automated transition refinement reduces method development time and increases data quality
• tMS3 acquisition enhances specificity, beneficial for complex biological samples

These features support applications in biomarker validation, signaling pathway studies, and quality control in pharmaceutical research.

Future Trends and Opportunities
Further work will assess assay performance at lower sample inputs (e.g., 10 µg) and additional reproducibility tests. The methodology may be extended to other post‐translational modifications and integrated into clinical proteomics workflows. Advances in instrument control software and acquisition strategies will likely drive even greater sensitivity and throughput.

Conclusion
The combination of PRM Conductor–optimized acquisition and a high‐speed nominal mass spectrometer delivers sensitive, reproducible quantitation of phosphopeptides. Both tMS2 and tMS3 approaches are feasible at discovery scale, with tMS3 offering enhanced specificity. This platform represents a significant improvement in assay throughput and sensitivity over traditional triple quadrupole methods.

References
1. Keshishian et al., Mol Syst. Biol., 17(9), 2021.
2. Abelin et al., Mol. Cell Proteomics, 15(5), 2016.