N-Terminomics: A High-Content Screen for Protease Substrates and Their Cleavage Sites
Proteases play vital roles in many cellular processes and signaling cascades through specific limited cleavage of their targets. It is important to identify what proteins are substrates of proteases and where their cleavage sites are so as to reveal the molecular mechanisms and specificity of signaling. We have developed a method to achieve this goal using a strategy that chemically tags the substrate’s alpha amine generated by proteolysis, enriches for tagged peptides, and identifies them using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Peptide MS/MS data are searched against a database to reveal what proteins are cleaved, whereby peptide N-termini demarcate sites of protease cleavage.
Key wordsCleavage site protease substrate N-terminomics biotin labeling signaling
We would like to thank Mari Enoksson, Wenhong Zhu, and Eric Wildfang for their effort and expertise, which was essential for the development of N-terminomics. This work was supported by the US National Institutes of Health (NIH) Roadmap Initiative National Biotechnology Resource Center grant RR20843 for the Center on Proteolytic Pathways, CA69381 from the National Cancer Institute (NCI), RPG0024/2006 from the Human Frontiers Science Program, and by Training Grant 5T32CA77109-9 from the NCI.