Unlocking the Proteome of Archival Tissue Collections : Proteomic Analysis of Urinary Bladder Cancer Tissues using Mass Spectrometry-based Proteomics

Abstract: Formalin-fixed and paraffin-embedded (FFPE) and optimal cutting temperature (OCT)-embedded frozen tissue samples have recently gained interest as they exist in vast repositories worldwide and represent a valuable resource for clinical studies. However, unlocking the proteome of these archival tissues has encountered many challenges. Formalin has deleterious effects on protein structure which has been a barrier to use such a tissue material for global discovery and post-translational modification (PTM) analysis using mass spectrometry (MS). Furthermore, a cryopreservation OCT medium composed of polymers, such polyvinyl alcohol and polyethylene glycol, interferes with MS analysis due to ion suppression effects. Recent developments in sample preparation protocols and MS-based proteomics have enabled to investigate these archived samples and opened up the possibility to integrate them into biomarker discovery. In this present study, I demonstrate a streamlined, compatible, reproducible, and high-throughput proteomic workflow for subsequent MS-based ‘shotgun’ and PTM analysis of FFPE and OCT-embedded frozen tissues using nanoLC-MS/MS. For the first time, the comprehensive comparison of protein expression in paired FFPE and OCT-embedded frozen tissues of invasive and non-muscle invasive bladder cancer is presented. Label-free quantification revealed more than 2,000 and 3,000 protein identities in FFPE and OCT-embedded frozen bladder cancer tissues, respectively. In total, 24 and 29 proteins showed differential protein expression in FFPE and OCT/frozen when comparing invasive stage versus non-muscle invasive bladder cancer. Exploring molecular interactions demonstrated that regulated proteins play a crucial role in bladder cancer-associated pathways. These preliminary results, therefore, reveal the potential to apply our optimized proteomic workflow for biomarker and drug target discovery. Furthermore, I tested titanium dioxide phosphopeptide enrichment on paired FFPE and OCT-embedded frozen tissue sample. More than three hundred phosphorylation sites were identified in both types of samples. This proteomic platform could also be a promising tool for PTM analysis in archival tissue samples.