Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories

Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource for retrospective clinical studies. Here, we evaluate the feasibility of (phospho-)proteomics on FFPE lung tissue regarding protein extraction, quantification, pre-analytics, and sample size. After comparing protein extraction protocols, we use the best-performing protocol for the acquisition of deep (phospho-)proteomes from lung squamous cell and adenocarcinoma with >8,000 quantified proteins and >14,000 phosphosites with a tandem mass tag (TMT) approach. With a microscaled approach, we quantify 7,000 phosphosites, enabling the analysis of FFPE biopsies with limited tissue amounts. We also investigate the influence of pre-analytical variables including fixation time and heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for the most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. Finally, we present general guidelines to which methods are best suited for different applications, highlighting TMT methods for comprehensive (phospho-)proteome profiling for focused clinical studies and label-free methods for large cohorts.