New prm-PASEF for highly multiplexed targeted acquisition in clinical samples

Significance of the Topic

The prm-PASEF approach addresses a critical need in targeted proteomics for high sensitivity and throughput. By integrating trapped ion mobility separation with parallel accumulation–serial fragmentation, this method enhances selectivity and multiplexing capacity. It is particularly relevant for clinical proteomics, where quantifying hundreds of peptide targets in limited sample amounts is essential for biomarker validation and large-scale studies.

Aims and Overview of the Study

This work introduces prm-PASEF as a novel acquisition strategy on the timsTOF Pro platform. The study aims to demonstrate its ability to measure multiple peptide precursors sequentially from a single ion mobility scan without sacrificing sensitivity. Performance metrics were evaluated using AQUA peptide standards spiked into a HeLa cell lysate, with future application intended for clinical samples.

Methodology and Instrumentation

The experiment employed nano-HPLC separation on a 250 mm pulled-emitter column with a 30 min gradient. Peptides were analyzed on a timsTOF Pro mass spectrometer operated in prm-PASEF mode.

• Chromatography: nanoElute system with IonOpticks columns.
• Mass spectrometry: Prototype prm-PASEF on the timsTOF Pro.
• Data analysis: Skyline-daily software for quantification.

Main Results and Discussion

The prm-PASEF method enabled selection of up to 11 peptide precursors within a single 100 ms ion mobility scan. A total of 215 precursors were targeted across a 30 min LC gradient, achieving:

• High sensitivity with limits of quantification in the low attomole range.
• Excellent accuracy (80–120% recovery) and precision (RSD < 5%) for calibration standards.
• Robust multiplexing, delivering consistent chromatographic peak profiles and over 10 data points per peak for reliable quantification.

Ion mobility filtering reduced chemical noise, while time-focusing effects improved fragment ion intensity. The approach maintained sensitivity despite high parallelization of targeting events.

Benefits and Practical Applications

prm-PASEF offers several advantages for analytical laboratories:

• Simultaneous quantification of hundreds of peptides in a single run.
• Improved selectivity through combined ion mobility and quadrupole isolation.
• Compatibility with high-throughput workflows for clinical and biomarker studies.

Future Trends and Potential Applications

Ongoing developments may extend prm-PASEF to routine clinical diagnostics, enabling large-cohort studies with minimal sample consumption. Integration with automated sample preparation and advanced data analysis pipelines could further boost throughput. Adapting the method to novel ion mobility designs and coupling with higher-resolution chromatography will likely enhance coverage and depth of proteome profiling.

Conclusion

This study validates prm-PASEF as a powerful targeted proteomics tool, combining high multiplexing, selectivity, and sensitivity. Its implementation on the timsTOF Pro platform paves the way for large-scale, quantitative analysis in biomedical research and clinical applications.