Identification of protease cleavage sites and substrates in cancer by carboxy-TAILS (C-TAILS)

Abstract

Determination of drug targets and development of novel therapeutics for the treatment of different cancers are actively ongoing areas of research. Proteases being the second largest group of enzymes in humans present themselves as attractive targets for blocking and activation to treat malignancies. However, determination of the protease cleavage substrates is often missed by utilizing conventional modern proteomic approaches. The relatively low abundance of proteolytically processed, and mostly semi-tryptic, peptides compared to tryptic peptides generated in shotgun proteomics compounded with their poorer identification rates makes the identification of such critical peptides challenging and so are mostly overlooked. Our laboratory introduced Terminal Amine Isotopic Labeling of Substrates (TAILS) to identify N-terminal peptides from cleavage events. In this chapter we present a protocol from our complementary method carboxy-TAILS (C-TAILS) to identify C-terminal peptides in metabolically labeled cancer cell lines.