Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)–MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.
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Research Article|
September 12 2007
Profiling constitutive proteolytic events in vivo
John C. Timmer;
John C. Timmer
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
†Graduate Program in Molecular Pathology, University of California, San Diego, La Jolla, CA 92093, U.S.A.
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Mari Enoksson;
Mari Enoksson
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Eric Wildfang;
Eric Wildfang
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Wenhong Zhu;
Wenhong Zhu
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Yoshinobu Igarashi;
Yoshinobu Igarashi
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Jean-Benard Denault;
Jean-Benard Denault
1
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Yuliang Ma;
Yuliang Ma
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Benjamin Dummitt;
Benjamin Dummitt
‡Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Boulevard, St. Louis, MO 63124, U.S.A.
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Yie-Hwa Chang;
Yie-Hwa Chang
‡Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Boulevard, St. Louis, MO 63124, U.S.A.
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Alan E. Mast;
Alan E. Mast
§Blood Center of Wisconsin, Milwaukee, WI 53201, U.S.A.
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Alexey Eroshkin;
Alexey Eroshkin
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Jeffrey W. Smith;
Jeffrey W. Smith
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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W. Andy Tao;
W. Andy Tao
∥Department of Biochemistry and Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, U.S.A.
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Guy S. Salvesen
Guy S. Salvesen
2
*Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
†Graduate Program in Molecular Pathology, University of California, San Diego, La Jolla, CA 92093, U.S.A.
2To whom correspondence should be addressed (email gsalvesen@burnham.org).
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Publisher: Portland Press Ltd
Received:
June 08 2007
Revision Received:
July 19 2007
Accepted:
July 24 2007
Accepted Manuscript online:
July 24 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 407 (1): 41–48.
Article history
Received:
June 08 2007
Revision Received:
July 19 2007
Accepted:
July 24 2007
Accepted Manuscript online:
July 24 2007
Connected Content
A commentary has been published:
Finding the needles in the haystack: mapping constitutive proteolytic events in vivo
Citation
John C. Timmer, Mari Enoksson, Eric Wildfang, Wenhong Zhu, Yoshinobu Igarashi, Jean-Benard Denault, Yuliang Ma, Benjamin Dummitt, Yie-Hwa Chang, Alan E. Mast, Alexey Eroshkin, Jeffrey W. Smith, W. Andy Tao, Guy S. Salvesen; Profiling constitutive proteolytic events in vivo. Biochem J 1 October 2007; 407 (1): 41–48. doi: https://doi.org/10.1042/BJ20070775
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