Gel-Free Proteomics pp 243-255 | Cite as
N-Terminomics: A High-Content Screen for Protease Substrates and Their Cleavage Sites
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 signalingNotes
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.
References
- 1.auf dem Keller, U., Doucet, A., and Overall, C. M. (2007) Protease research in the era of systems biology, Biol Chem 388, 1159–1162.PubMedCrossRefGoogle Scholar
- 2.Fuentes-Prior, P., and Salvesen, G. S. (2004) The protein structures that shape caspase activity, specificity, activation and inhibition, Biochem J 384, 201–232.PubMedCrossRefGoogle Scholar
- 3.Timmer, J. C., and Salvesen, G. S. (2007) Caspase substrates, Cell Death Differ 14, 66–72.PubMedCrossRefGoogle Scholar
- 4.Timmer, J. C., Enoksson, M., Wildfang, E., Zhu, W., Igarashi, Y., Denault, J. B., Ma, Y., Dummitt, B., Chang, Y. H., Mast, A. E., Eroshkin, A., Smith, J., Tao, W. A., and Salvesen, G. S. (2007) Profiling constitutive proteolytic events in vivo, Biochem J 407, 41–48.PubMedCrossRefGoogle Scholar
- 5.Timmer, J. C., Zhu, W., Pop, C., Regan, T., Snipas, S. J., Eroshkin, A. M., Riedl, S. J., and Salvesen, G. S. (2009) Structural and kinetic determinants of protease substrates, Nat Struct Mol Biol 16, 1101–1108.PubMedCrossRefGoogle Scholar
- 6.Elias, J. E., and Gygi, S. P. (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry, Nat Methods 4, 207–214.PubMedCrossRefGoogle Scholar
- 7.Crooks, G. E., Hon, G., Chandonia, J. M., and Brenner, S. E. (2004) WebLogo: a sequence logo generator, Genome Res 14, 1188–1190.PubMedCrossRefGoogle Scholar
- 8.Vacic, V., Iakoucheva, L. M., and Radivojac, P. (2006) Two sample logo: a graphical representation of the differences between two sets of sequence alignments, Bioinformatics (Oxford, England) 22, 1536–1537.CrossRefGoogle Scholar
- 9.Elias, J. E., Haas, W., Faherty, B. K., and Gygi, S. P. (2005) Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations, Nat Methods 2, 667–675.PubMedCrossRefGoogle Scholar