N-Terminomics: A High-Content Screen for Protease Substrates and Their Cleavage Sites

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 753)

Abstract

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 words

Cleavage site protease substrate N-terminomics biotin labeling signaling 

Notes

Acknowledgments

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.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of PharmacologyUniversity of California San DiegoLa JollaUSA
  2. 2.Program in Apoptosis and Cell Death Research, Sanford-Burnham Medical Research InstituteLa JollaUSA

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