Proteolytic Fingerprinting of Complex Biological Samples Using Combinatorial Libraries of Fluorogenic Probes

Kalyani Jambunathan1, Douglas S. Watson1, Krishna Kodukula1, Amit K. Galande1

1 Center for Advanced Drug Research, Biosciences Division, SRI International, Harrisonburg, Virginia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.22
DOI:  10.1002/0471140864.ps2122s70
Online Posting Date:  November, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Proteases have garnered interest as candidate biomarkers and therapeutic targets for many human diseases. A key challenge is the identification and characterization of disease‐relevant proteases in the complex milieu of biological fluids such as serum, plasma, and bronchoalveolar lavage, in which a multitude of hydrolases act in concert. This unit describes a protocol to map the global proteolytic substrate specificities of complex biological samples using a concise combinatorial library of internally quenched fluorogenic peptide probes (IQFPs). This substrate profiling approach provides a global and quantitative comparison of protease specificities between different biological samples. Such a comparative analysis can lead to the identification of disease‐specific ‘fingerprints' of proteolytic activities with potential utility in diagnosis and therapy. Curr. Protoc. Protein Sci. 70:21.22.1‐21.22.14. © 2012 by John Wiley & Sons, Inc.

Keywords: FRET; global proteolytic specificities; proteases; protease substrate identification

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Optimization of Protease Assay Screen with FITC‐Casein
  • Alternate Protocol 1: Optimization of Protease Assay Screen with Generic Peptide Probes
  • Basic Protocol 2: IQFP Library Screen of Biological Samples
  • Basic Protocol 3: Deconvolution of IQFP Peptide Motifs Identified in the Library Screen
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Optimization of Protease Assay Screen with FITC‐Casein

  Materials
  • Biological sample(s)—culture supernatant, serum, plasma or BAL
  • Serine protease buffer (see recipe)
  • Metalloprotease buffer (see recipe)
  • Cysteine protease buffer (see recipe)
  • Aspartic protease buffer (see recipe)
  • 100 µM fluorescein isothiocyanate–labeled casein (FITC‐casein; Anaspec) in H 2O
  • 0.5 mg/ml sequencing‐grade modified trypsin, porcine (Promega, cat. no. V5113)
  • 10% (w/v) trichloroacetic acid
  • 50 mM HEPES buffer, pH 8.0
  • 1.7‐ml microcentrifuge tubes (Eppendorf)
  • 5‐ to 50‐µl 8‐channel pipettor with tips
  • 1‐ to 10‐µl 8‐channel pipettor with tips
  • Eppendorf (or equivalent) tabletop centrifuge capable of 21,000 × g
  • 96‐well low‐volume black microtiter plates (Molecular Devices)
  • Analyst HT plate reader (Molecular Devices) or any other plate reader with time resolved fluorescence capability and appropriate instrument software
  • Excitation and emission fluorescence filters (485 nm/530 nm for FITC‐Casein; 320 nm/420 nm for MCA/DNP)
  • Spreadsheet software (e.g., Microsoft Excel)

Alternate Protocol 1: Optimization of Protease Assay Screen with Generic Peptide Probes

  • IQFP Probe MCA‐RPPGFSAFK (DNP) (Anaspec; see Table 21.22.1 for other peptides)

Basic Protocol 2: IQFP Library Screen of Biological Samples

  Materials
  • 50 nmol REPLi library (Mimotopes; see recipe for IQFP library stock solutions)
  • 1:1 acetonitrile:water
  • Serine protease buffer (see recipe)
  • Metalloprotease buffer (see recipe)
  • Cysteine protease buffer (see recipe)
  • Aspartic protease buffer (see recipe)
  • Appropriately diluted biological sample
  • 96‐well low‐volume black microtiter plates (Molecular Devices)
  • 5‐ to 50‐µl 8‐channel pipettor with tips
  • 1‐ to 10‐µl 8‐channel pipettor with tips
  • 1.7‐ml microcentrifuge tubes (Eppendorf)
  • Seal & Sample aluminum foil lids (Beckman Coulter, cat. no. 538619)
  • Analyst HT plate reader (Molecular Devices) or any other plate reader with time resolved fluorescence capability and appropriate instrument software
  • Excitation and emission fluorescence filters (485 nm/530 nm for FITC‐Casein; 320 nm/420 nm for MCA/DNP)
  • Spreadsheet software (e.g., Microsoft Excel)
  • Heat map builder, e.g., from the Euan Ashley Lab at Stanford University (King et al., )

Basic Protocol 3: Deconvolution of IQFP Peptide Motifs Identified in the Library Screen

  Materials
  • Individual IQFP peptides identified from library screen ( protocol 3)
  • Dimethylsulfoxide (DMSO), molecular biology grade
  • Assay buffer used in the library screen (one of the following):
    • Serine protease buffer (see recipe)
    • Metalloprotease buffer (see recipe)
    • Cysteine protease buffer (see recipe)
    • Aspartic protease buffer (see recipe)
  • Appropriately diluted biological sample
  • 1.7‐ml microcentrifuge tubes (Eppendorf)
  • 96‐well low‐volume black microtiter plates (Molecular Devices)
  • 5‐ to 50‐µl 8‐channel pipettor with tips
  • 1‐ to 10‐µl 8‐channel pipettor with tips
  • Seal & Sample aluminum foil lids (Beckman Coulter, cat. no. 538619)
  • Analyst HT plate reader (Molecular Devices) or any other plate reader with time resolved fluorescence capability and appropriate instrument software
  • Excitation and emission fluorescence filters (485 nm/530 nm for FITC‐Casein; 320 nm/420 nm for MCA/DNP)
  • Spreadsheet software (e.g., Microsoft Excel)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Backes, B.J., Harris, J.L., Leonetti, F., Craik, C.S., and Ellman, J.A. 2000. Synthesis of positional‐scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat. Biotechnol. 18:187‐193.
   Cravatt, B.F., Simon, B.M., and Yates, J.R. III. 2007. The biological impact of mass spectrometry‐based proteomics. Nature 450:991‐1000.
   Cravatt, B.F., Wright, A.T., and Kozarich, J.W. 2008. Activity‐based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77:383‐414.
   Diamandis, E.P. 2004. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool: Opportunities and potential limitations. Mol. Cell. Proteomics 3:367‐378.
   King, J.Y., Ferrara, R., Tabibiazar, R., Spin, J.M., Chen, M.M., Kuchinsky, A., Vailaya, A., Kincaid, R., Tsalenko, A., Deng, D.X., Connolly, A., Zhang, P., Yang, E., Watt, C., Yakhini, Z., Ben‐Dor, A., Adler, A., Bruhn, L., Tsao, P., Quertermous, T., and Ashley, E.A. 2005. Pathway analysis of coronary atherosclerosis. Physiol. Genomics 23:103‐118.
   Thomas, D.A., Francis, P., Smith, C., Ratcliffe, S., Ede, N.J., Kay, C., Wayne, G., Marin, S.L., Moore, K., Amour, A., and Hooper, N.M. 2006. A broad spectrum fluorescence based peptide library for the rapid identification of protease substrates. Proteomics 6:2112‐2120.
   Watson, D.S., Jambunathan, K., Askew, D.S., Kodukula, K., and Galande, A.K. 2011a. Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases. BioTechniques 51:95‐104.
   Watson, D.S., Feng, X., Askew, D.S., Jambunathan, K., Kodukula, K., and Galande, A.K. 2011b. Substrate specificity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr. PLoS One 6:1‐16.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library