Abstract 2039: Confident analyses of proteins in FFPE tissue: Robust, reliable, reproducible sample preparation enables LC-MS based proteomics assays
Authors: O'Hare Tom , Daviso Eugenio , Bhattacharyya Debadeep , Vasantgadkar Sameer 
Published: June 2022

Abstract
Formalin-fixed paraffin-embedded (FFPE) tissue samples are routinely used in clinical histopathology. While FFPE preserves tissue architecture, removal of paraffin is an essential to achieve high quality LC-MS data. Xylol, despite all its efficacy presents severe health risks and xylol removal compromises assay reproducibility. Superior sample preparation ensures removal of unwanted matrix components and provides reliability, reproducibility and robustness of the assays. This is achieved by an optimal combination of lysis by Adaptive Focused Acoustics (AFA®) with a modified protein aggregation capture (PAC) protocol. This method offers robust, reliable, and reproducible separation of paraffin from the tissue and ensures efficient purification of protein. FFPE scrolls were used in a Covaris plate, incubated post-treatment with Tissue Lysis Buffer, and processed with the Covaris LE220-Plus Focused-ultrasonicator for deparaffinization and DNA shearing. PAC protocol was used to extract from FFPE lysate with additional washing steps to ensure paraffin removal. Sera Mag Speed beads (bead:protein 1:5) were used to induce on-bead protein aggregation followed by washing and resuspension in 100 mM Tris-HCl (pH ~ 8) for digestion with Trypsin and LysC. All samples were measured on a 100 min gradient using a Q Exactive HF-X Orbitrap High Resolution Accurate Mass Spectrometer coupled to an EASY-nLC 1200 UHPLC. FFPE scrolls of human adenoma samples were used and processed in single wells. An optimal combination of heating and AFA ensured reversal of crosslinks and paraffin removal from tissue samples. Protein purification was achieved using the PAC protocol. Finally, a tryptic ‘on-bead’ digest was performed resulting in a clean peptide solution that was desalted prior to LC-MS analysis. A comparison of the xylol-free workflow with that of the common xylol deparaffinization resulted in highly similar peptide, protein peptide identification rates, and protein abundance ranges. On average, 3,900 proteins and 20,842 peptides per single run measurement were quantified. From the perspective of monitoring post-translational modifications, a comparison of this method with the xylol-based protocol showed that crosslink reversal was equally efficient in both protocols. Efficiency of tissue lysis and paraffin removal in relation to the thickness of tissue sections were evaluated in 3 and 10 µm thick adenoma sections. While both section types provided enough material for multiple MS measurements, 10 µm thick sections showed a higher spread of peptide yields. The number of protein identifications, however, was found to be similar under both conditions. To further assess the reproducibility of the AFA and PAC workflow, three tissue sections of a colorectal adenoma were used, resulting in quantification of more than 3,500 proteins in each of the tissue replicates.
Citation Format: Tom O'Hare, Eugenio Daviso, Debadeep Bhattacharyya, Sameer Vasantgadkar. Confident analyses of proteins in FFPE tissue: Robust, reliable, reproducible sample preparation enables LC-MS based proteomics assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2039.