An Optimized Sample Preparation Method of Formalin-Fixed Paraffin-Embedded Tissues for Mass Spec Applications

Significance of the Topic

The analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by mass spectrometry (MS) unlocks decades of archived clinical samples for proteomic research. Standardizing protein extraction from FFPE materials is critical to generate reproducible, high-coverage datasets that support biomarker discovery, disease profiling, and translational studies.

Study Objectives and Overview

This work presents an optimized workflow to extract, digest, label, fractionate, and analyze proteins from FFPE tissues. Key aims were to compare paraffin removal and lysis conditions across breast, lung, and colon samples, validate compatibility with Thermo Scientific EasyPep sample-prep kits, employ TMT10plex isobaric tagging for quantitative analysis, and benchmark results against fresh frozen counterparts.

Methodology

The protocol comprises four main stages:

• Paraffin removal and rehydration of FFPE curls (1–2 h).
• Lysis with EasyPep buffer, micro-pestle homogenization plus probe sonication, and heat-induced crosslink reversal (95 °C, 2 h).
• Protein quantification (Rapid-Gold BCA), enzymatic digestion with EasyPep Mini MS kit, and peptide assay normalization.
• TMT10plex labeling, quenching, pooling, high-pH reversed-phase fractionation, and LC-MS/MS on a Q Exactive HF instrument.

Used Instrumentation

• Thermo Scientific Dionex Ultimate 3000 Nano LC with 50 cm EASY-Spray C18 column.
• Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap MS.
• Thermo Scientific EasyPep Mini MS Sample Prep Kit and TMT10plex reagents.
• Pierce High pH Reversed-Phase Peptide Fractionation spin columns.

Main Results and Discussion

The optimized lysis approach yielded 2,000–3,500 proteins per FFPE sample, with EasyPep buffer and micro-pestle homogenization outperforming standard methods. TMT10plex analysis quantified over 5,000 proteins across five tissue pairs, demonstrating robust relative quantification. Comparison to fresh frozen samples showed similar protein identification numbers (>4,400 proteins), although volcano‐plot analyses revealed sample-specific abundance shifts in tumors vs. normal tissues.

Practical Benefits and Applications

• Enables high-throughput proteomic profiling of archived clinical specimens.
• Integrates seamlessly with established Thermo Scientific workflows.
• Supports quantitative differential expression studies in oncology and pathology.

Future Trends and Applications

Advancements may include fully automated FFPE processing platforms, improved crosslink reversal chemistries, deeper proteome coverage via extended LC gradients, integration with single-cell proteomics, and AI-driven data interpretation to accelerate biomarker discovery.

Conclusion

This optimized FFPE sample-prep method delivers high protein yields, broad proteome coverage, and reliable quantitative performance. It leverages EasyPep kits and TMT10plex labeling to unlock the potential of archived tissue collections for diverse research and clinical applications.