Peptidomic Approaches to Study Proteolytic Activity

Peter J. Lyons1, Lloyd D. Fricker1

1 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.13
DOI:  10.1002/0471140864.ps1813s65
Online Posting Date:  August, 2011
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Peptidomics, the analysis of the peptide content of cells or tissues, can be used to study proteases in several ways. First, nearly all of the peptides detected in cells and tissues are proteolytic fragments of proteins. Analysis of the peptides therefore provides information regarding the proteolytic activities that occurred to generate the observed peptides. The use of quantitative peptidomic approaches allows the comparison of relative peptide levels in two or more different samples, which enables studies examining the consequences of increasing proteolytic activity (by enzyme activation or overexpression) or reducing proteolytic activity (by inhibition, knock down, or knock out). Quantitative peptidomics can also be used to directly test the cleavage specificity of purified proteases. For this, peptides are purified from the tissue or cell line of interest, incubated in the presence of various amounts of protease or in the absence of protease, and then analyzed by the quantitative peptidomics approach. This reveals which peptides are preferred substrates, which are products, and which are not cleaved. Collectively, these studies complement conventional approaches to study proteolytic activity and allow for a more complete understanding of an enzyme's substrate specificity. This unit describes the use of quantitative peptidomics in the analysis of the biological peptidome as well as in the in vitro analysis of peptidase activity. Curr. Protoc. Protein Sci. 65:18.13.1‐18.13.12. © 2011 by John Wiley & Sons, Inc.

Keywords: proteomics; protease; mass spectrometry

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation and Analysis of Peptides by LC/MS
  • Basic Protocol 2: Peptidomic Analysis of LC/MS Data
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation and Analysis of Peptides by LC/MS

  • Mice
  • 0.1 M hydrochloric acid (from 6 N, sequanal grade, constant boiling, Pierce)
  • 0.4 M NaH 2PO 4 (Sigma‐Aldrich)
  • TMAB‐NHS compounds (synthesis of these compounds has been described in Morano et al., )
  • 1.0 M NaOH (Sigma‐Aldrich)
  • Dimethyl sulfoxide (Sigma‐Aldrich)
  • NH 2OH⋅HCl (Sigma‐Aldrich)
  • Glycine (Sigma‐Aldrich)
  • Acetonitrile, HPLC grade (Fisher Scientific)
  • Trifluoroacetic acid (Pierce)
  • Formic acid
  • Low‐retention microcentrifuge tubes (Eppendorf)
  • Milli‐Q distilled water system (Millipore)
  • Ultrasonic processor (e.g., model W‐380, Ultrasonic Inc.)
  • Hydrion pH papers, 8.0‐9.5 (Micro Essential Laboratory)
  • Centrifugal filter devices (Amicon Ultra 4‐ml Ultracel, 10,000 MWCO, Millipore)
  • PepClean C‐18 spin column (Pierce)
  • LC/MS with C18 trapping column (e.g., Symmetry C18 trapping column, 5‐µm particles, 180‐µm i.d. × 20 mm, Waters) and separating column (e.g., BEH 130 C18 column, 1.7‐µm particles, 100‐µm i.d. × 100 mm, Waters)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.
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Literature Cited

Literature Cited
   Berezniuk, I., Sironi, J., Callaway, M.B., Castro, L.M., Hirata, I.Y., Ferro, E.S., and Fricker, L.D. 2010. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24:1813‐1823.
   Berti, D.A., Morano, C., Russo, L.C., Castro, L.M., Cunha, F.M., Zhang, X., Sironi, J., Klitzke, C.F., Ferro, E.S., and Fricker, L.D. 2009. Analysis of intracellular substrates and products of thimet oligopeptidase (EC in human embryonic kidney 293 cells. J. Biol. Chem. 284:14105‐14116.
   Che, F.Y. and Fricker, L.D. 2005. Quantitative peptidomics of mouse pituitary: Comparison of different stable isotopic tags. J. Mass Spectrom. 40:238‐249.
   Ferro, E.S., Hyslop, S., and Camargo, A.C. 2004. Intracellullar peptides as putative natural regulators of protein interactions. J. Neurochem. 91:769‐777.
   Fricker, L.D. 2010. Analysis of mouse brain peptides using mass spectrometry‐based peptidomics: Implications for novel functions ranging from non‐classical neuropeptides to microproteins. Mol. Biosyst. 6:1355‐1365.
   Fricker, L.D., Lim, J., Pan, H., and Che, F.Y. 2006. Peptidomics: Identification and quantification of endogenous peptides in neuroendocrine tissues. Mass Spectrom. Rev. 25:327‐344.
   Gelman, J.S., Sironi, J., Castro, L.M., Ferro, E.S., and Fricker, L.D. 2010. Hemopressins and other hemoglobin‐derived peptides in mouse brain: Comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice. J. Neurochem. 113:871‐880.
   Gelman, J.S., Wardman, J.H., Bhat, V.B., Gozzo, F.C., and Fricker, L.D. In Press. Quantitative peptidomics to measure neuropeptide levels in animal models relevant to psychiatric disorders. In Psychiatric Disorders: Methods and Protocols (F. Kobeissy, ed.). Humana Press.
   Julka, S. and Regnier, F. 2004. Quantification in proteomics through stable isotope coding: A review. J. Proteome Res. 3:350‐363.
   Lyons, P.J. and Fricker, L.D. 2010. Substrate specificity of human carboxypeptidase A6. J. Biol. Chem. 285:38234‐38242.
   Mahrus, S., Trinidad, J.C., Barkan, D.T., Sali, A., Burlingame, A.L., and Wells, J.A. 2008. Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134:866‐876.
   Morano, C., Zhang, X., and Fricker, L.D. 2008. Multiple isotopic labels for quantitative mass spectrometry. Anal. Chem. 80:9298‐9309.
   Svensson, M., Skold, K., Svenningsson, P., and Andren, P.E. 2003. Peptidomics‐based discovery of novel neuropeptides. J. Proteome Res. 2:213‐219.
   Tanco, S., Zhang, X., Morano, C., Aviles, F.X., Lorenzo, J., and Fricker, L.D. 2010. Characterization of the substrate specificity of human carboxypeptidase A4 and implications for a role in extracellular peptide processing. J. Biol. Chem. 285:18385‐18396.
   Wardman, J.H., Zhang, X., Gagnon, S., Castro, L.M., Zhu, X., Steiner, D.F., Day, R., and Fricker, L.D. 2010. Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics. J. Neurochem. 114:215‐225.
   Zhang, R., Sioma, C.S., Thompson, R.A., Xiong, L., and Regnier, F.E. 2002. Controlling deuterium isotope effects in comparative proteomics. Anal. Chem. 74:3662‐3669.
   Zhang, X., Pan, H., Peng, B., Steiner, D.F., Pintar, J.E., and Fricker, L.D. 2010. Neuropeptidomic analysis establishes a major role for prohormone convertase‐2 in neuropeptide biosynthesis. J. Neurochem. 112:1168‐1179.
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