The human genome encodes more than 560 proteases, but only for few of them the substrate proteins are known. This is mainly due to high numbers of potential substrate targets for any given protease that cannot be comprehensively explored by conventional candidate approaches. In this chapter, we describe a proteomics protocol for the reliable identification of protease substrates on a proteome-wide scale. Notably, this method termed iTRAQ-Terminal Amine Isotopic Labeling of Substrates (TAILS) does not require any prior knowledge on candidate proteins. Instead, it is used as an unbiased discovery approach to identify protease-substrate relations in complex biological samples. In addition, iTRAQ-TAILS not only identifies substrate proteins but also maps cleavage sites with amino acid precision. Knowing the cleavage site enables the researcher to perform specific downstream analyses and eliminates the need for laborious follow-up experiments like Edman sequencing. iTRAQ-TAILS acquires this rich information by exploiting the power of latest generation mass spectrometers as the Thermo Q Exactive instrument. Through quantitative assessment of protein N-termini in protease-exposed and control samples and a robust data analysis pipeline, iTRAQ-TAILS can systematically screen proteome-wide substrate spaces for proteolytic events exerted by proteases of interest.
- Protease substrate screening
- Thermo Q Exactive