Deep, Reproducible and High throughput FFPE Analyses: Moving Toward Large Scale Clinical Omics Applications

Significance of the Topic

Formalin fixation and paraffin embedding (FFPE) is the global standard for tissue preservation in pathology. These samples form massive archives with linked clinical data and represent an underutilized resource for proteomic omics research. Advancing high-throughput, reproducible workflows for FFPE analysis is essential to unlock retrospective studies and support large-scale clinical decision-making.

Study Objectives and Overview

This work integrates the ProtiFi HYPERsol sample processing protocol with high-speed data acquisition on the Bruker timsTOF Pro to enable same-day preparation and analysis of 50 FFPE samples. The goal was to achieve deep proteome coverage, low variability, and full automation in a 24-hour workflow.

Methodology and Instrumentation

Mouse liver FFPE cores were processed in 96-well plates by dissolving in 5% SDS, heat-induced reverse crosslinking at 85 °C for two hours, and ultrasonication using a PIXUL device. Proteins were cleaned and digested on S-Trap microplates with trypsin. Peptides were separated using a 20-minute gradient on a 15 cm C18 column in a nanoElute LC system and analyzed by data-dependent PASEF on the timsTOF Pro. Data processing employed PEAKS X+ and Simplifi software.

Instrumentation Used

• PIXUL 96-well ultrasonicator
• S-Trap microplates (ProtiFi)
• nanoElute nanoLC system (Bruker Daltonics)
• 15 cm C18 1.6 μm column (Ionopticks)
• timsTOF Pro mass spectrometer (Bruker Daltonics) with dda-PASEF
• PEAKS X+ and Simplifi data analysis suites

Key Results and Discussion

The HYPERsol workflow delivered high-yield protein extraction and recovery directly from FFPE material. Chromatographic peak widths averaged 2.1 s with sustained high sampling rates (>70 precursors/s). Across 50 samples processed in one day, an average of 3 540 protein groups and 17 757 unique peptides were identified with coefficients of variation below 4%. Pairwise correlation of label-free quantitation signals yielded an average Pearson r of 0.981, demonstrating exceptional reproducibility.

Benefits and Practical Applications

• Automatable sample processing compatible with 96-well formats accelerates large-scale studies.
• Short LC gradients combined with trapped ion mobility offer excellent depth without long run times.
• High reproducibility and throughput support translational proteomics and retrospective biomarker discovery.

Future Trends and Potential Applications

Integration of this streamlined FFPE workflow into clinical research pipelines will enable multi-site cohort studies, longitudinal disease monitoring, and integration with genomics and histopathology data. Further advances in instrument speed and sample processing miniaturization will increase throughput beyond 100 samples per day.

Conclusion

The combination of HYPERsol processing and timsTOF Pro dda-PASEF acquisition provides a robust, high-throughput solution for deep and reproducible proteomic profiling of FFPE tissues in under 24 hours. This platform paves the way for large-scale clinical omics and retrospective studies leveraging vast FFPE archives.

References

1. J. Proteome Res. 2020, 19, 2, 973–98
2. Nucleic Acids Res. 2019, 47, 12