prm-PASEF®: enabling high-throughput, high sensitivity targeted proteomics

Importance of the topic

• Targeted proteomics provides high sensitivity, specificity and reproducibility for hypothesis‐driven studies and biomarker verification.
• Conventional SRM and PRM methods face trade‐offs between the number of targets, chromatographic duration and sensitivity.
• The prm‐PASEF strategy leverages trapped ion mobility to overcome these limitations and increase throughput without loss of performance.

Objectives and study overview

• Introduce and evaluate the prm‐PASEF acquisition method on a timsTOF Pro instrument.
• Assess multiplexing capability, sensitivity, quantification accuracy and reproducibility using a HeLa digest spiked with 201 heavy AQUA peptides and 15 light standards.

Instrumentation

• nanoElute UHPLC system with 250 mm IonOpticks pulled‐emitter column.
• timsTOF Pro mass spectrometer operated in prm‐PASEF mode.
• Data processing performed in Skyline‐daily (version 20.1.1.83).

Methodology

• Sample preparation: 100 ng/µL HeLa digest spiked with 201 heavy AQUA peptides; 15 heavy/light pairs prepared in a nine‐point dilution series (5.5 to 50 000 amol/µL).
• Chromatography: 30 min gradient from 2 % to 30 % acetonitrile at nanoflow rates.
• prm‐PASEF settings: 50 ms accumulation, 100 ms separation; mobility range 0.65–1.3 1/K₀; m/z range 100–1700.
• Quantification metrics: limits of quantification defined by 80–120 % accuracy and signal > mean(blank)+3×SD; reproducibility by relative standard deviation (RSD) over triplicates.

Key results and discussion

• Multiplexing: 216 precursors monitored in a single 30 min run with an average of 4 targets per PASEF frame and up to 10 frames per cycle.
• Sensitivity: LOQs down to 17.2 amol achieved for select peptides; linear dynamic range spanning over three orders of magnitude with 80–120 % accuracy.
• Reproducibility: median of 25 data points per chromatographic peak and RSD values below 5 % without internal standard normalization.
• Ion mobility separation minimized overlap in three‐dimensional acquisition windows, preserving selectivity and sensitivity even under high target density.

Benefits and practical applications

• Enables high‐throughput targeted assays with short gradients (potentially ≤5 min) without compromising data completeness.
• Ideal for large‐scale clinical studies requiring robust quantification of peptide biomarkers.
• Compatible with fast LC/UHPLC setups and high target load, reducing instrument time per sample.

Future trends and applications

• Integration with ultrafast LC methods (e.g., ≤5 min gradients) for rapid screening workflows.
• Expansion to even larger target panels by exploiting advanced ion mobility and parallel accumulation strategies.
• Potential combination with data‐independent approaches and automated data analysis pipelines for comprehensive proteome coverage.

Conclusion

• prm‐PASEF on timsTOF Pro combines high multiplexing, sensitivity, speed and selectivity in a single method.
• Demonstrates accurate quantification, excellent reproducibility and flexibility for short and high‐throughput targeted proteomics applications.

References

• Lesur A., Schmit P.-O., Adams C., Dittmar G. prm‐PASEF: enabling high‐throughput, high sensitivity targeted proteomics. Bruker Daltonics Application Note TN-52, 10/2020.