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

In this study, we evaluated the feasibility of proteomics and phosphoproteomics on formalin-fixed paraffin-embedded (FFPE) lung tissue for protein extraction protocols, quantification methods, pre-analytics, and sample size. First, we compared three protocols for the extraction of proteins from FFPE tissues and used the best-performing protocol to acquire a deep proteome of lung adenocarcinoma and squamous cell carcinoma. We quantified >8,000 proteins and >14,000 phosphosites with a tandem mass tag (TMT11) approach and 6,700 proteins and 7,000 phosphosites with a microscaled TMT approach, enabling the analysis of FFPE needle biopsies with limited amounts of tissue. This is a considerable increase in coverage compared to label-free quantification techniques. We also evaluated pre-analytical variables such as the influence of fixation times and the duration of heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. We also present general guidelines to what proteomics and phosphoproteomics methods are best suited for different applications for retrospective cancer studies.