Abstract
Formalin-fixed paraffin-embedded (FFPE) samples are valuable archived bio-specimens of individuals and are commonly used in biomedical research. Here, we present a protocol for deep proteomic profiling of FFPE specimens using a spectral library-free approach. We describe steps for FFPE tissue collection, tissue lysis, homogenization, protein lysate cleanup, on-beads digestion, and de-salting. We then detail data acquisition and statistical analysis. This protocol is highly sensitive, reproducible, and applicable for high-throughput proteomic profiling and can be used on various types of specimens.