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Gel-Free Proteomics

Volume 753 of the series Methods in Molecular Biology pp 243-255

Date:

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

  • John C. TimmerAffiliated withDepartment of Pharmacology, University of California San Diego
  • , Guy S. SalvesenAffiliated withProgram in Apoptosis and Cell Death Research, Sanford-Burnham Medical Research Institute Email author 

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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