A streamlined technique for in-depth ubiquitomics analysis of formalin-fixed paraffin-embedded tissues

Formalin-fixed paraffin-embedded (FFPE) tissues offer advantages over fresh frozen (FrFr) tissues in terms of preserving tissue integrity, reducing long-term storage costs, and increasing sample availability, thus providing an invaluable resource for biomarker discovery and exploration of disease mechanisms. As a reversible and highly versatile post-translational modification (PTM), ubiquitination is involved in protein homeostasis and the regulation of almost all physiological and pathological processes, but ubiquitomics studies of FFPE tissues are lacking due to technical challenges. In this study, we proposed an optimized, streamlined technique for extracting GlyGly proteins from FFPE tissues, including improvements in the components of lysis buffer, the duration of heating for tissue sections and the strategy for protein digestion. The application of this strategy to clinical samples revealed that 7000-10 000 GlyGly sites could be identified from several FFPE tissue sections of hepatocellular carcinoma (HCC). Finally, we showed that the storage duration had a minor effect on the stability of ubiquitomics information within 7 years, while phosphoproteomics information remained stable for more than 10 years. Our strategy proves that FFPE tissues can serve as a valuable alternative to FrFr tissues for comprehensive ubiquitomics investigations.