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PMID: 17650073 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Validation Study

Profiling constitutive proteolytic events in vivo.

The Biochemical journal ·Vol. 407 ·No. 1 ·2007-10-01 ·Pages 41-8

Timmer JC, Enoksson M, Wildfang E, Zhu W, Igarashi Y, Denault JB, Ma Y, Dummitt B, Chang YH, Mast AE, Eroshkin A, Smith JW, Tao WA, Salvesen GS

Abstract

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.

MeSH Terms
Amino Acid Sequence Aminopeptidases/chemistry,metabolism Animals Aprotinin/chemistry,metabolism Blood Proteins/chemistry,metabolism Caspases/chemistry,metabolism Cells, Cultured Chromatography, Liquid/methods Genome, Fungal Humans Methionyl Aminopeptidases Methylurea Compounds/pharmacology Mice Mitochondria/metabolism Models, Biological Molecular Sequence Data Peptide Hydrolases/metabolism Peptides/analysis,chemistry,metabolism Proteome/analysis,metabolism Proteomics/methods Tandem Mass Spectrometry/methods
Chemicals
Blood Proteins Methylurea Compounds Peptides Proteome O-methylisourea Aprotinin Peptide Hydrolases Aminopeptidases Methionyl Aminopeptidases Caspases
Authors & Affiliations
14 authors, click to expand affiliations / ORCID
Timmer John C
Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Enoksson Mari
Wildfang Eric
Zhu Wenhong
Igarashi Yoshinobu
Denault Jean-Benard
Ma Yuliang
Dummitt Benjamin
Chang Yie-Hwa
Mast Alan E
Eroshkin Alexey
Smith Jeffrey W
Tao W Andy
Salvesen Guy S
References (35)
35 references, click to expand
  1. Mitochondrial processing peptidase: multiple-site recognition of precursor proteins.
    Biochem Biophys Res Commun. 1999 Nov 30;265(3):611-6 PMID: 10600469
  2. Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12.
    Electrophoresis. 1997 Aug;18(8):1259-313 PMID: 9298646
  3. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries.
    Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7754-9 PMID: 10869434
  4. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.
    Biochem J. 2000 Sep 1;350 Pt 2:563-8 PMID: 10947972
  5. Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library.
    J Biol Chem. 2000 Oct 6;275(40):31422-7 PMID: 10906330
  6. Caspases: intracellular signaling by proteolysis.
    Cell. 1997 Nov 14;91(4):443-6 PMID: 9390553
  7. N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families.
    Trends Biochem Sci. 1998 Jul;23(7):263-7 PMID: 9697417
  8. Fibroblast activation protein-alpha and dipeptidyl peptidase IV (CD26): cell-surface proteases that activate cell signaling and are potential targets for cancer therapy.
    Drug Resist Updat. 2005 Feb-Apr;8(1-2):51-8 PMID: 15939342
  9. Investigating diversity in human plasma proteins.
    Proc Natl Acad Sci U S A. 2005 Aug 2;102(31):10852-7 PMID: 16043703
  10. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations.
    Nat Methods. 2005 Sep;2(9):667-75 PMID: 16118637
  11. Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis.
    Nat Methods. 2005 Oct;2(10):771-7 PMID: 16179924
  12. Positional proteomics: selective recovery and analysis of N-terminal proteolytic peptides.
    Nat Methods. 2005 Dec;2(12):955-7 PMID: 16299481
  13. MEROPS: the peptidase database.
    Nucleic Acids Res. 2006 Jan 1;34(Database issue):D270-2 PMID: 16381862
  14. Degradomics: systems biology of the protease web. Pleiotropic roles of MMPs in cancer.
    Cancer Metastasis Rev. 2006 Mar;25(1):69-75 PMID: 16680573
  15. Guanidination chemistry for qualitative and quantitative proteomics.
    Rapid Commun Mass Spectrom. 2006;20(21):3245-56 PMID: 17019669
  16. The proteomics of N-terminal methionine cleavage.
    Mol Cell Proteomics. 2006 Dec;5(12):2336-49 PMID: 16963780
  17. Identification of proteolytic cleavage sites by quantitative proteomics.
    J Proteome Res. 2007 Jul;6(7):2850-8 PMID: 17547438
  18. Enhancing the intensities of lysine-terminated tryptic peptide ions in matrix-assisted laser desorption/ionization mass spectrometry.
    Rapid Commun Mass Spectrom. 2000;14(23):2147-53 PMID: 11114023
  19. Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors.
    Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):2967-72 PMID: 11248015
  20. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries.
    Nat Biotechnol. 2001 Jul;19(7):661-7 PMID: 11433279
  21. Protease degradomics: a new challenge for proteomics.
    Nat Rev Mol Cell Biol. 2002 Jul;3(7):509-19 PMID: 12094217
  22. Mitochondrial processing peptidases.
    Biochim Biophys Acta. 2002 Sep 2;1592(1):63-77 PMID: 12191769
  23. Signal peptidases.
    Chem Rev. 2002 Dec;102(12):4549-80 PMID: 12475201
  24. N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins.
    J Mol Biol. 2003 Jan 24;325(4):595-622 PMID: 12507466
  25. Factors governing nonoverlapping substrate specificity by mitochondrial inner membrane peptidase.
    J Biol Chem. 2003 Feb 14;278(7):4943-8 PMID: 12482857
  26. Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry identify age-related protein expression differences for the primary visual cortex of kitten and adult cat.
    J Neurochem. 2003 Apr;85(1):193-205 PMID: 12641741
  27. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.
    Nat Biotechnol. 2003 May;21(5):566-9 PMID: 12665801
  28. Dipeptidyl peptidase IV inhibitors for the treatment of impaired glucose tolerance and type 2 diabetes.
    Curr Opin Investig Drugs. 2003 Apr;4(4):412-20 PMID: 12808880
  29. Improved prediction of signal peptides: SignalP 3.0.
    J Mol Biol. 2004 Jul 16;340(4):783-95 PMID: 15223320
  30. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid.
    Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247-51 PMID: 2682640
  31. Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth.
    Mol Gen Genet. 1995 Jan 20;246(2):247-53 PMID: 7862096
  32. Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries.
    J Biol Chem. 1995 Mar 24;270(12):6440-9 PMID: 7896777
  33. Origins of the specificity of tissue-type plasminogen activator.
    Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7627-31 PMID: 7644467
  34. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.
    J Biol Chem. 1997 Jul 18;272(29):17907-11 PMID: 9218414
  35. Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases.
    J Neurosci. 1997 Aug 15;17(16):6165-78 PMID: 9236228
Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
1470-8728
DOI
10.1042/BJ20070775
Published
2007-10-01
Pages
41-8
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC2267409
Subset
IM
Grants
NCRR NIH HHS · R21 RR019752 · United States
NCRR NIH HHS · U54 RR020843 · United States
NCRR NIH HHS · RR19752 · United States
NCRR NIH HHS · RR20843 · United States
Corrections
CommentIn
PMID: 17822382
External Links
PubMed source DOI: 10.1042/BJ20070775 PMC Full Text View journal details
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