Automated and faster library-free dia-PASEF analysis with a Spectronaut integrated workflow in ProteoScape

Significance of the Topic

Recent advances in high-throughput proteomics have driven a shift towards faster chromatographic workflows and library-free data-independent acquisition (DIA) techniques, notably dia-PASEF on timsTOF instruments. This approach enables deep coverage of complex samples with minimal analysis times, addressing the growing demand for large-cohort studies in both basic research and clinical applications. Streamlined processing pipelines that integrate acquisition and analysis workflows are critical to maximize throughput and ensure data quality.

Objectives and Study Overview

This study evaluates the integration of Spectronaut’s directDIA+ library-free analysis module within Bruker ProteoScape (BPS) to achieve automated, real-time processing of dia-PASEF data. The aim is to compare speed, identification performance, and quantitation accuracy between the BPS integrated workflow and the standalone Spectronaut v18 application using two datasets: a multi-species mix with known ratios and a phosphorylation-enriched sample set.

Methodology and Instruments

• Data Acquisition: dia-PASEF on timsTOF Pro with a 45 min reversed-phase LC gradient and 15 min overhead, two samples with five technical replicates each.
• Workflow Comparison: BPS integrated Spectronaut directDIA+ (single injection real-time processing with project-level combine) vs standalone Spectronaut v18 after full file acquisition.
• Datasets: a multi-species human/yeast/E.coli mixture (ratios 1:1:1, 1:2:4) for precision/accuracy assessment; a phosphopeptide-enriched dataset from Skowronek et al. (2022).
• Data Analysis Metrics: protein group, protein, precursor, peptide, and modified sequence identification counts; quantitative ratio accuracy; coefficient of variation (CV); phosphopeptide overlap and PTM localization confidence.

Main Results and Discussion

The integrated BPS pipeline reduced total processing time by approximately 40% (2.5 h faster) compared to the traditional method, thanks to concurrent data streaming and on-the-fly analysis. Identification numbers for proteins, precursors, peptides, and modified sequences were highly comparable between workflows, with CVs and quantitative ratios matching expected values. Phosphopeptide analysis exhibited over 90% overlap between BPS and standalone results with a localization filter ≥0.75, demonstrating robust PTM site assignment. The integrated environment also allowed immediate run-by-run quality checks, enabling early detection of suboptimal injections.

Benefits and Practical Applications

• Significant reduction in time-to-results facilitates large-scale proteomics projects and high-throughput screening.
• Unified platform streamlines the workflow, minimizing manual file transfers and batch configuration steps.
• Real-time run-level feedback allows dynamic decision making, improving data quality and instrument utilization.
• Library-free directDIA+ ensures broad proteome coverage without the need for extensive DDA spectral libraries.

Future Trends and Opportunities

Future developments may focus on further parallelization within the BPS processing engine, enhanced PTM site localization algorithms, and incorporation of emerging AI-driven identification tools. Expanding support for additional sample preparation strategies, deeper integration with laboratory information management systems, and adaptive real-time acquisition adjustments will drive next-generation proteomics throughput and reproducibility.

Conclusion

The integration of Spectronaut directDIA+ within Bruker ProteoScape offers an automated, high-throughput solution for library-free dia-PASEF analysis, reducing processing time by nearly half while maintaining identification depth and quantitative accuracy. This fully integrated workflow empowers proteomics laboratories to scale up studies efficiently without compromising data integrity.

References

• Lopez J, Brehmer S, Skoraczynski G, Abuin J, Gandhi T, Bernhardt O, Reiter L, Trede D, Krieger J, Srikumar T. Automated and faster library-free dia-PASEF analysis with a Spectronaut integrated workflow in ProteoScape. US HUPO 2024, Abstract 663.