Formalin-fixed and paraffin-embedded (FFPE) and optimal cutting temperature (OCT)-embedded and frozen tissue specimens in biobanks are highly valuable in clinical studies but proteomic and post-translational modification (PTM) studies using mass spectrometry (MS) have been limited due to structural arrangement of proteins and contaminations from embedding material. This study aims to develop a parallel proteomic workflow for FFPE and OCT/frozen samples that allows for large-scale, quick, reproducible qualitative and quantitative high-resolution MS-analysis. The optimized protocol gives details on removal of embedding material, protein extraction, and multi-enzyme digestion using filter-aided sample preparation method. The method was evaluated by investigating the protein expression levels in non-muscle-invasive and muscle-invasive bladder cancer samples in two cohorts. More than 2000 and 3000 proteins in FFPE and OCT/frozen samples, respectively, were identified, and samples could be clustered in different tumour stages based on their protein expression. Furthermore, more than 250 and 400 phosphopeptides could be identified from specific patient samples of FFPE and OCT/frozen, respectively, using titanium dioxide enrichment. The paper presents unique data describing the similarities and differences observed in FFPE and OCT/frozen samples and shows the feasibility to detect proteins and site-specific phosphorylation even after long-term storage of clinical samples.
Analytical chemistry. 2018 Apr 06 [Epub ahead of print]
Ales Holfeld, Alberto Valdés, Per-Uno Malmström, Ulrika Segersten, Sara Bergstrom Lind