Session: Session 2

Unlocking the proteomic potential of FFPE tissues with BeatBox® and iST: A xylene-free, high-throughput workflow for in-depth proteome analysis

Objective

Formalin-fixed, paraffin-embedded (FFPE) tissue despite being accessible as widely used in routine clinical diagnostics remain extremely challenging for proteomics analysis as harsh conditions are required to remove cross-links and paraffin. By combining the BeatBox® tissue homogenizer with an optimized iST workflow, it is now possible to process 96 FFPE samples per day easily, efficiently, and reproducibly without using toxic xylene-based deparaffinization opening up a new avenue in proteomics for biomarker discovery.

 

Method

FFPE curls (10 µm, non-deparaffinized) from mouse cardiac muscle, kidney, and liver with matching snap-frozen tissue (1-2 mg) were processed in 96-well format. For FFPE samples, an optimized workflow was established: Homogenization in the BeatBox® (high power, 10 min), followed by an one-hour incubation at 80-95 °C, to de-crosslink, extract, reduce and alkylate proteins. Applying the iST sample preparation protocol, tryptic digestion was followed by an optimized peptide clean-up to remove last traces of paraffin. Peptides were analyzed on a nano-LC coupled to a timsTOF mass spectrometer using diaPASEF® acquisition mode. Raw files were analyzed using DIA-NN or Bruker ProteoScapeTM.

 

Results

This novel xylene-free FFPE workflow using BeatBox® and iST was compared to a traditional workflow with xylene-based deparaffinization and bead-based sonication. For all tested tissues, BeatBox homogenization revealed an increase in protein IDs of >10% and up to 43% for mouse cardiac muscle while achieving an excellent intra- and interday repeatability with median CVs of <10% (n=4). Comparing FFPE tissues with the corresponding fresh-frozen tissues revealed more than 10000 proteins for both conditions (fresh-frozen vs. FFPE) with up to 73% common proteins and a similar dynamic range.

 

Conclusion

Thanks to this innovative high-throughput workflow, retrospective clinical proteomic studies for biomarker discovery from FFPE tissue can now become routine work.