Specificity Profiling of Protein‐Binding Domains Using One‐Bead‐One‐Compound Peptide Libraries

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

One?bead?one?compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331?355 © 2012 by John Wiley & Sons, Inc.

Keywords: protein?protein interaction; protein binding domains; sequence specificity; peptide libraries; one?bead?one?compound libraries; SH2 domains

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Introduction
Strategic Planning
Basic Protocol 1: Split‐and‐Pool Synthesis of One‐Bead‐One‐Compound Libraries
Alternate Protocol 1: Synthesis of an Inverted Peptide Library
Basic Protocol 2: Chemical Labeling of Proteins with NHS‐Biotin
Alternate Protocol 2: Enzymatic Labeling of Proteins with Biotin‐CoA
Basic Protocol 3: On‐Bead Screening of One‐Bead‐One‐Compound Libraries
Alternate Protocol 3: On‐Bead Screening Against Fluorescently Labeled Protein
Basic Protocol 4: Partial Edman Degradation for Sequencing Support‐Bound Peptides
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Split‐and‐Pool Synthesis of One‐Bead‐One‐Compound Libraries

Materials

TentaGel S NH₂ resin (90 µm, 0.3 mmol/g)
Dichloromethane (DCM; Sigma Aldrich)
Diethyl ether
Fmoc‐Met‐OSu (Aapptech, http://www.aapptec.com)
Boc‐Phe‐OSu (Aapptech, http://www.aapptec.com)
N ′,N ′‐dimethylformamide (DMF; Sigma‐Aldrich)
Acetaldehyde
Chloranil (Sigma‐Aldrich, cat. no. 45374)
Fmoc‐Met‐OH (Aapptech, http://www.aapptec.com)
O ‐Benzotriazole‐N ,N ,N ′,N ′‐tetramethyluronium hexafluorophosphate (HBTU; Aapptech, http://www.aapptec.com)
N ‐Hydroxybenzotriazole (HOBt; Aapptech, http://www.aapptec.com)
Diisopropylethylamine (DIPEA)
Piperidine
All desired Fmoc‐amino acids with acid‐liable sidechain protecting groups (tBu, Boc, etc.; Aapptech, http://www.aapptec.com)
Ninhydrin
Ethanol
Phenol
Potassium cyanide (KCN)
CD₃ CO₂ D (Sigma‐Aldrich)
CH₃ CD₂ CO₂ D (Sigma‐Aldrich)
Fmoc‐pY‐OH (Aapptech)
Acetic anhydride
Modified reagent K (see recipe )

Chemglass solid‐phase peptide‐synthesis vessel with inner luer joint, >25 ml; Disc O.D: 20 mm; GL size: 25 (CG186202, Chemglass, cat. no. CG‐1862‐02)
Rotary shaker
Spatula
Watch glass
Vacuum source
50‐ml conical centrifuge tubes (BD Falcon)

Alternate Protocol 1: Synthesis of an Inverted Peptide Library

Nα‐Fmoc‐Glu(δ‐N ‐hydroxysuccinimidyl)‐O‐CH₂ CH=CH₂ (Aapptech, http://www.aapptec.com)
Boc‐Gly‐OH (Aapptech, http://www.aapptec.com)
p ‐Hydroxymethylbenzoic acid (HMBA; Sigma‐Aldrich)
Trifluoroacetic acid (TFA)
Fmoc‐Arg(Pbf)‐OH (Aapptech, http://www.aapptec.com)
N,N‐dicyclohexylcarbodiimide (DCC; Sigma‐Aldrich)
4‐Dimethylaminopyridine (DMAP)
Tetrakis(triphenylphosphine)palladium (Sigma‐Aldrich)
Triphenylphosphine
N‐Methylaniline (Sigma‐Aldrich)
0.5% sodium dimethyldithiocarbamate hydrate (Sigma‐Aldrich)
Benzotriazole‐1‐yl‐oxy‐tris‐pyrrolidino‐phosphonium hexafluorophosphate (PyBOP; Aapptech, http://www.aapptec.com)
1 M NaOH

Basic Protocol 2: Chemical Labeling of Proteins with NHS‐Biotin

Materials

Purified protein of interest dissolved in any buffer without strong nucleophiles (avoid Tris)
Bradford assay kit (BioRad)
Bicarbonate buffer: 100 mM sodium bicarbonate/100 mM NaCl
NHS‐biotin stock solution: dissolve NHS‐biotin (Thermo Scientific, cat. no. 21312) in DMSO for a final concentration of 10 mg/ml (store in small aliquots up to 1 year at −20 or −80°C
Sephadex G‐25 resin (GE Healthcare; optional)
Protein purification buffer: 50 mM HEPES/100 mM NaCl; adjust pH to 7.5 with HCl or NaOH
30% or 50% (v/v) glycerol

Amicon ultracentrifugation unit (Millipore)
Bio‐Spin disposable chromatography columns (BioRad, cat. no. 732‐6008; optional)

Alternate Protocol 2: Enzymatic Labeling of Proteins with Biotin‐CoA

Materials

50 to 500 µM protein to be labeled in any common buffer (avoid metal chelators such as EDTA)
10× Sfp reaction buffer (see recipe )
Purified Sfp enzyme (Yin et al., )
Coenzyme A–biotin: Dissolve biotin‐CoA (New England Biolabs, cat. no. S59351S) in DMSO to a final concentration of 10 mg/ml and store it in small aliquots at −20° or −80°C.
Protein purification buffer: 50 mM HEPES/100 mM NaCl; adjust pH to 7.5 with HCl or NaOH
Sephadex G‐25 column (GE Healthcare)

Basic Protocol 3: On‐Bead Screening of One‐Bead‐One‐Compound Libraries

Materials

Library resin (synthesized in protocol 1 )
N ′,N ′‐dimethylformamide (DMF; Sigma Aldrich)
Dichloromethane (DCM; Sigma Aldrich)
HBST blocking buffer (see recipe )
Biotinylated protein ( protocol 3 )
SAAP buffer (see recipe )
1 mg/ml SAAP (streptavidin–alkaline phosphatase; Prozyme, http://www.prozyme.com/)
Staining buffer (see recipe )
5 mg/ml BCIP in H₂ O (Sigma‐Aldrich)
1 N HCl

Micro‐BioSpin columns (0.8 ml; BioRad)
Rotary shaker
35‐mm Petri dish (nonsterile, non‐culture‐treated polystyrene dishes work fine)
Dissection microscope (10× to 40× magnification)

Alternate Protocol 3: On‐Bead Screening Against Fluorescently Labeled Protein

Materials

Library resin (synthesized on PEGA resin in protocol 1 )
N ′,N ′‐dimethylformamide (DMF; Sigma Aldrich)
HBST blocking buffer (see recipe )
Fluorescently labeled protein (e.g., protocol 3 )

Micro‐BioSpin columns (0.8 ml; BioRad
Rotary shaker
Fluorescence microscope

Basic Protocol 4: Partial Edman Degradation for Sequencing Support‐Bound Peptides

Materials

Positive beads ( protocol 5 or protocol 6 )
Pyridine
Triethylamine
8.8 to 11.0 mM fluorenylmethyoxylcarbonyl‐N‐hydroxysuccinimidyl ester (Fmoc‐OSu; Sigma‐Aldrich) in pyridine
Phenylisothiocyanate (PITC; can be purchased in 1‐ml sealed ampules from Sigma‐Aldrich)
Dichloromethane (DCM)
20% (v/v) piperidine in dimethylformamide (DMF)
Trifluoroacetic acid (TFA; Sigma‐Aldrich)
Dimethyl sulfide (Sigma‐Aldrich)
Ammonium iodide
40 mg/ml cyanogen bromide in 70% (v/v) TFA
Acetonitrile
Methanol
4‐hydroxy‐α‐cyanocinnamic (Sigma‐Aldrich)

Custom‐designed reaction vessel (12‐mm diameter, 20 mm height, a 10‐ 20‐µm glass frit, and 1 mm luer tip at the bottom; see Fig. )
Speedvac evaporator and vacuum source

Additional reagents and equipment for MALDI‐TOF mass spectrometry (Henzel and Stults, )

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Figures

Figure 1. (A ) Scheme showing the main steps in the spatial segregation of TentaGel beads and synthesis of an inverted peptide library containing free C‐termini. (B ) Picture of spatially segregated beads from (A) after staining of the N‐terminal amines of peptides with chloranil (under a microscope and stained prior to side chain deprotection).

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Figure 2. Scheme showing bead segregation and the reduction of surface ligand density. Reagents and conditions: (a) soak in water overnight; (b) 0.4 equivalents Boc‐Phe‐OSu, 0.1 equivalent Fmoc‐Met‐OSu, and 0.5 equivalent DIPEA in 55:45 diethyl ether/DCM, 30 min; (c) 4 equivalents Fmoc‐Met‐OH, HBTU; (d) 55% TFA in DCM; (e) acetic anhydride; (f) split‐and‐pool synthesis by Fmoc/HBTU chemistry; (g) TFA.

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Figure 3. Scheme showing the reactions involved in the colorimetric library screening of a pY peptide library against biotinylated GST‐SH2 domains. In this figure, a bead (the large black circle) has a peptide which is binding through its pY (the triangle with a P in it, to an SH2 domain. The SH2 domain is a fusion with a GST, which has been labeled chemically with biotin. During screening, streptavadin–alkaline phosphatase (SAAP) is added, which binds to the biotin and causes a change in color on the bead. The resulting bead is blue, and can be picked out with a pipettor and pipet tip and sequenced.

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Figure 4. (A ) Reactions involved in partial Edman degradation. Reagents and conditions: (a) 10‐30:1 PITC:Fmoc‐OSu; (b) TFA; (c) Repeat steps a and b seven times; (d) 20% piperidine in DMF; and (e) CNBr in 70% TFA. (B ) A representative MALDI‐TOF spectrum showing the peptide ladder derived from a single bead that displays the peptide sequence AHIpYDFIYLNBBRM‐resin.

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Figure 5. Custom‐designed reaction vessel.

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Videos

Literature Cited

Literature Cited
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Specificity Profiling of Protein‐Binding Domains Using One‐Bead‐One‐Compound Peptide Libraries

Abstract

Table of Contents

Materials

Basic Protocol 1: Split‐and‐Pool Synthesis of One‐Bead‐One‐Compound Libraries

Alternate Protocol 1: Synthesis of an Inverted Peptide Library

Basic Protocol 2: Chemical Labeling of Proteins with NHS‐Biotin

Alternate Protocol 2: Enzymatic Labeling of Proteins with Biotin‐CoA

Basic Protocol 3: On‐Bead Screening of One‐Bead‐One‐Compound Libraries

Alternate Protocol 3: On‐Bead Screening Against Fluorescently Labeled Protein

Basic Protocol 4: Partial Edman Degradation for Sequencing Support‐Bound Peptides

Figures

Videos

Literature Cited