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Identification of Proteolytic Products and Natural Protein N-Termini by Terminal Amine Isotopic Labeling of Substrates (TAILS)

  • Alain Doucet
  • Oded Kleifeld
  • Jayachandran N. Kizhakkedathu
  • Christopher M. OverallEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 753)

Abstract

Determining the sequence of protein N-termini and their modifications functionally annotates proteins since translation isoforms, posttranslational modifications, and proteolytic truncations direct localization, activity, and the half-life of most proteins. Here we present in detail the steps required to perform our recently described approach we call Terminal Amine Isotopic Labeling of Substrates (TAILS), a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative and global analysis of the N-terminome and proteolysis in one MS/MS experiment. By a 3-day procedure with flexible α- and ɛ-amine labeling and blocking options, TAILS removes internal tryptic and C-terminal peptides by binding to a dendritic polyglycerol aldehyde polymer. Therefore, by negative selection, this enriches for both the N-terminal-labeled peptides and all forms of naturally blocked N-terminal peptides. In addition to providing valuable proteome annotation, the simultaneous analysis of the original mature N-terminal peptides enables these peptides to be used for higher confidence protein substrate identification by two or more different and unique peptides. Second, the analysis of the N-terminal peptides forms a statistical classifier to determine valid isotope ratio cutoffs in order to identify with high-confidence protease-generated neo-N-terminal peptides. Third, quantifying the loss of acetylated or cyclized N-terminal peptides that have been cleaved extends overall substrate coverage. Hence, TAILS allows for the global analysis of the N-terminome and determination of cleavage site motifs and substrates for protease including those with unknown or broad specificity.

Key words

Protein N-termini N-terminome degradomics N-terminomics functional proteomics proteolysis protease substrates acetylated proteins 

Notes

Acknowledgments

A.D. acknowledges the Fonds Quebecois de la Recherche sur la Nature et les Technologies and the Michael Smith Foundation for Health Research (MSFHR) for research fellowships. O.K. acknowledge the Centre for Blood Research for a postdoctoral fellowship. C.M.O. is supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology. J.N.K. is a recipient of a CIHR/Canadian Blood Services new investigator award in transfusion science. This work was supported by grants from the CIHR and from a program project grant in Breast Cancer Metastases from the Canadian Breast Cancer Research Alliance with funds from the Canadian Breast Cancer Foundation and The Cancer Research Society as well as with an Infrastructure Grant from the Canada Foundation for Innovation (CFI) and MSHFR to the UBC Centre of Blood Research. We thank Dr. Wei Chen and the CBR mass spectrometry core facility, and Dr. Leonard Foster for proteomic analysis and Dr. Georgina Butler for critical reading and editing of the manuscript.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Alain Doucet
    • 1
    • 4
  • Oded Kleifeld
    • 2
    • 5
  • Jayachandran N. Kizhakkedathu
    • 1
  • Christopher M. Overall
    • 3
    Email author
  1. 1.Department of Pathology and Laboratory MedecineCentre for Blood Research, University of British ColumbiaVancouverCanada
  2. 2.Department of Biochemistry and Molecular BiologyCentre for Blood Research, University of British ColumbiaVancouverCanada
  3. 3.Department of Biochemistry and Molecular Biology, Department of Oral Biological and Medical SciencesCentre for Blood Research, University of British ColumbiaVancouverCanada
  4. 4.Department of Biochemistry, Microbiology and ImmunologyOttawa Institute of Systems Biology, University of OttawaOttawaCanada
  5. 5.Faculty of Biology, Israel Institute of TechnologyHeifaIsrael

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