Proteomic techniques and activity-based probes for the system-wide study of proteolysis

doi:10.1016/j.biochi.2010.04.027

Biochimie

Volume 92, Issue 11, November 2010, Pages 1705-1714

https://doi.org/10.1016/j.biochi.2010.04.027 Get rights and content

Abstract

Proteolysis constitutes a major post-translational modification but specificity and substrate selectivity of numerous proteases have remained elusive. In this review, we highlight how advanced techniques in the areas of proteomics and activity-based probes can be used to investigate i) protease active site specificity; ii) protease in vivo substrates; iii) protease contribution to proteome homeostasis and composition; and iv) detection and localization of active proteases. Peptide libraries together with genetical or biochemical selection have traditionally been used for active site profiling of proteases. These are now complemented by proteome-derived peptide libraries that simultaneously determine prime and non-prime specificity and characterize subsite cooperativity. Cell-contextual discovery of protease substrates is rendered possible by techniques that isolate and quantitate protein termini. Here, a novel approach termed Terminal Amine Isotopic Labeling of Substrates (TAILS) provides an integrated platform for substrate discovery and appropriate statistical evaluation of terminal peptide identification and quantification. Proteolytically generated carboxy-termini can now also be analyzed on a proteome-wide level. Proteolytic regulation of proteome composition is monitored by quantitative proteomic approaches employing stable isotope coding or label free quantification. Activity-based probes specifically recognize active proteases. In proteomic screens, they can be used to detect and quantitate proteolytic activity while their application in cellular histology allows to locate proteolytic activity in situ. Activity-based probes – especially in conjunction with positron emission tomography – are also promising tools to monitor proteolytic activities on an organism-wide basis with a focus on in vivo tumor imaging. Together, this array of methodological possibilities enables unveiling physiological protease substrate repertoires and defining protease function in the cellular- and organism-wide context.

Section snippets

Degradomics

Proteolytic enzymes sculpt proteomes through limited, site-specific processing or degradation of proteineous substrates. With over 550 proteases in man, the size of this enzyme class reflects the involvement of proteolysis in almost all physiological processes [1]. For most proteases, the in vivo substrate profiles remain to be elucidated. Limited knowledge of protease substrate repertoires restricts our understanding of protease biology and ultimately hinders to fully exploit the therapeutic

Active site specificity

Numerous experimental strategies have been developed to characterize the active site specificity of proteolytic enzymes. An overview of the methods discussed below is shown in Table 1.

Overview

Mass spectrometry together with gel-based or gel-free separation methods offers a powerful way for the rapid identification of 100s–1000s of proteins in biological systems. These techniques can also determine differential protein abundance in different samples. In gel-based systems, spot-staining intensity serves as a direct measure for protein abundance. In gel-free systems, samples are typically labeled with stable isotopes (such as the commercially available ICAT [23], iTRAQ [24] or SILAC

Overview

Global quantitative comparison of different proteomes is inadequate for the determination of proteolytic cleavage sites in complex biological samples as well as being insufficient to profile subtle changes caused by limited, site-specific proteolysis. Since proteolysis creates neo protein termini, multiple strategies have been designed with the aim to monitor proteolytic events on a proteome-wide scale by identifying and quantifying protein N-termini. Generally, these methods enrich for

Overview

Most proteases are tightly regulated on the post-translational level and aberrant protease activities are characteristics of diseases such as cancer and arthritis. However, techniques monitoring protein abundances do not account for these changes and while giving insight into mechanisms of protease action the identification of substrates and cleavage sites does not detect subtle but detrimental alterations in activity levels. Furthermore, those techniques are based on the analysis of protein

Perspective

No single method alone can uncover the multiple roles of proteases in physiology and pathology. In this review we have highlighted how degradomic techniques offer new routes to uncover key features in protease biology, including specificity, proteome-wide effects, natural substrates, and localization of proteolytic activity. An integrated, multi-facetted approach that appropriately combines these technologies will ultimately provide the best platform to characterize and understand proteolysis

Acknowledgement

O.S. acknowledges support from the Deutsche Forschungsgemeinschaft (DFG) (grant SCHI871/2-1).

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ReviewProteomic techniques and activity-based probes for the system-wide study of proteolysis

Abstract

Section snippets

Degradomics

Active site specificity

Overview

Overview

Overview

Perspective

Acknowledgement

Curr. Opin. Chem. Biol.

Mol. Cell. Proteomics

Chem. Biol.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Chem. Biol.

J. Biol. Chem.

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Biochim. Biophys. Acta

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Cell

Anal. Biochem

Anal. Biochem.

Anal. Biochem.

Cell

Cell

Mol. Cell. Proteomics

Mol. Cell. Proteomics

Biochim. Biophys. Acta

Anal. Biochem.

Mol. Cell. Proteomics

J. Biol. Chem.

Curr. Opin. Genet. Dev.

Mol. Cell

Cancer Cell

Nucl. Med. Biol.

Nucl. Med. Biol.

Nucl. Med. Biol.

Human and mouse proteases: a comparative genomic approach

Nat. Rev. Genet.

Matrix metalloproteinase inhibitors and cancer: trials and tribulations

Science

Targeting proteases: successes, failures and future prospects

Nat. Rev. Drug Discov.

Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites

Nat. Biotechnol.

Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites

Mol. Biotechnol.

Protease degradomics: a new challenge for proteomics

Nat. Rev. Mol. Cell. Biol.

Substrate phage: selection of protease substrates by monovalent phage display

Science

Methods for mapping protease specificity

Curr. Opin. Chem. Biol.

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

Proc. Natl. Acad. Sci. U.S.A.

Peptide microarrays for the determination of protease substrate specificity

J. Am. Chem. Soc.

Rapid identification of substrates for novel proteases using a combinatorial peptide library

J. Comb. Chem.

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

Proteomics

Determination of protease cleavage site motifs using mixture-based oriented peptide libraries

Nat. Biotechnol.

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

Nat. Biotechnol.

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Nat. Biotechnol.

Identification of caspase-3 degradome by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis

Proteomics

A proteomic approach for the discovery of protease substrates

Proc. Natl. Acad. Sci. U.S.A.

Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis

Mol. Cell. Biol.

Review
Proteomic techniques and activity-based probes for the system-wide study of proteolysis