Oral Presentation 24th Annual Lorne Proteomics Symposium 2019

High-throughput proteomic analysis of FFPE tissue using PCT-SWATH MS (#56)

Steven G Williams 1 , Natasha Lucas 1 , Andrew B Robinson 1 , Belinda Serafin 1 , Peter G Hains 1 , Phil J Robinson 1
  1. The Childrens Medical Research Institute, Westmead, NSW, Australia

Formalin-fixed paraffin-embedded (FFPE) tissue archives present as a potential rich source for proteome data, spanning a vast range of diseases and time. While many methods for handling FFPE samples are known, they are limited by the large number of washing steps and tissue disintegration during preparation, and require up to 12 hours to achieve.  These issues limit use of these samples to small cohorts.  Currently, there is no published fast and reproducible method suitable for both high-throughput and small-scale sample analysis. Developing an effective method to analyse and collect proteomes from FFPE tissue would allow true high-throughput sample preparation that is robotics-capable to capitalise on the available samples.  This aim aligns with ProCan’s goal to process 70,000 tumour samples over the next 7 years. Here we describe an effective high-throughput protocol for the rapid processing of FFPE samples, including wax removal and cross-link reversal.  The method is based on pressure cycling technology (PCT) in a barocycler instrument. The washing steps are reduced to 3 minutes and changes to the barocycler processing allows compatibility with standardised workflows used for fresh frozen samples.  With this method, 96 samples can be taken from tissue sample to MS-ready in 6 hours. We outline the method optimisation and show that PCT is a robust and reproducible method with significant advances over traditional methods used to process archival tissue. By comparing the proteome by SWATH-mass spectrometry of several rat tissue FFPE samples to their equivalent fresh frozen samples, we show the variation between sample types was no different to that between samples of the same type. The results allow authentic high throughput sample preparation of FFPE tissues for the first time, suitable for robotics and industrial-scale proteomics analysis.