Gel-Free Proteomics pp 243-255

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

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

Protocol

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 

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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