Single-shot quantitative plasma proteomics using the PASEF method

Significance of the topic

The human plasma proteome is a critical resource for biological and clinical research but presents a vast dynamic range of protein concentrations that challenges in-depth, high-throughput analysis. The PASEF (parallel accumulation–serial fragmentation) approach on the timsTOF Pro platform enhances sequencing speed, ion utilization, and sensitivity, offering a streamlined workflow for large-cohort plasma studies without extensive depletion or fractionation.

Objectives and Study Overview

This study evaluated single-shot, label-free plasma proteomics using the PASEF method. Specific aims were to optimize the ion mobility range, assess different chromatographic gradient lengths, and explore library-based matching strategies to maximize protein identifications in undepleted plasma samples.

Methodology

• Sample preparation: Pooled human plasma was digested with trypsin and cleaned using PreOmics iST kits.
• Depletion workflow: Selective removal of abundant proteins via Agilent Human-6 and Sigma IgY14 columns, followed by iST digestion.
• Chromatography: 200 ng peptide loads on a 25 cm Aurora C18 column for 30–45 min gradients; a Bruker TEN column was used for 20 min gradients.
• Data acquisition: Data-dependent PASEF in oTOF control software for DDA runs; 25 min dia-PASEF experiments also performed.
• Data analysis: DDA raw files processed in MaxQuant (1% FDR, matching between runs); Spectronaut (Biognosys) used for dia-PASEF quantification.

Instrumentation Used

• nanoElute UHPLC system with Aurora 25 cm C18 and Bruker TEN columns
• timsTOF Pro mass spectrometer (Bruker Daltonics)
• oTOF control software, MaxQuant, Spectronaut for data processing

Main Results and Discussion

• Optimized ion mobility range (0.85–1.35 1/K0) yielded the highest protein identifications.
• In undepleted plasma, single-shot DDA PASEF achieved an average of 345 protein groups with a 45 min gradient, and ~280 with 20 min.
• Matching 30 min DDA runs to a depletion-based library increased identifications to >550 proteins (default matching) and >460 proteins under stringent retention (±6 s) and mobility (±0.01 1/K0) windows.
• Library-assisted 25 min dia-PASEF quantified >410 proteins in undepleted samples.
• Quantitative completeness exceeded 90% for most proteins across replicates; LFQ CV remained low for medium-to-high abundance targets.

Benefits and Practical Applications

• Enables rapid, deep profiling of large plasma cohorts without costly depletion or extensive fractionation.
• Streamlined label-free workflow reduces preparation time and cost.
• Flexible balance between throughput and depth suits both discovery and routine QC applications.

Future Trends and Potential Applications

• Longer chromatographic gradients may approach identification of ~1000 plasma proteins in single runs.
• Development of comprehensive spectral libraries or adoption of library-free DIA workflows to further boost coverage.
• Application to biomarker discovery, clinical diagnostics, and expansive cohort studies for translational research.

Conclusion

Single-shot PASEF acquisition on the timsTOF Pro provides a fast, sensitive, and reproducible strategy for plasma proteome profiling. Library-based matching significantly enhances depth, making this approach ideal for high-throughput biological and clinical investigations.