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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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

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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

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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 H₂ O
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)

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Figures

Figure 21.22.1 Endpoint fluorescence measurement for cleavage of FITC‐casein by Aspergillus fumigatus culture supernatant diluted 1:50 in assay buffers representing each of the four major protease classes. Maximum proteolytic activity was observed in both serine protease buffer (HEPES, pH 8.0) and metalloprotease buffer (Tris, pH 7.4). These experimental results, along with literature indicating that serine proteases are the most abundant class of proteases secreted in AF culture supernatant, led to the selection of HEPES buffer, pH 8.0, for IQFP library screens.

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Figure 21.22.2 Fluorescence fold change values observed for cleavage of the generic IQFP MCA‐RPPGFSAFK (DNP) by human plasma samples collected from the same patient at two different disease‐stage time points. The plasma samples were diluted 1:1 in four assay buffers. Maximum proteolytic activity for both plasma samples was observed in HEPES buffer, pH 8.0, which was then chosen for library screens of the plasma samples.

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Figure 21.22.3 IQFP library screen of pooled complement‐preserved normal human serum at 1:10 dilution. (A ) Heat map generated from fluorescence fold change values calculated at the assay end point ( t _6h ). The heat map corresponds to stacked 96‐well library plates as indicated. Each yellow square corresponds to a library well and the brightness of each square corresponds to calculated fold change value for that well. (B ) Example of IQFP library peptide containing the variable amino acid sequence of ‘AIL.’ (C ) Variable position amino acid distribution profile for library sequences cleaved by human serum.

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Figure 21.22.4 Deconvolution profile for the peptide motif I/L‐F/Y‐F/Y selected from the IQFP library screen of guinea pig BAL. (A ) A representation of a 96‐well assay plate with cleaved wells highlighted in green. The selected peptide motif is written in single‐letter amino acid code. (B ) Guinea pig BAL cleavage profile of the 8 individual constituent peptides from the motif. A fluorescence fold change value of 2 or higher is considered indicative of peptide cleavage (a ‘positive hit’).

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Literature Cited

Literature Cited
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