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

Andrew R. Kunys1, Wenlong Lian1, Dehua Pei1

1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120125
Online Posting Date:  December, 2012
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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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Table of Contents

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

  • TentaGel S NH 2 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)
  • CD 3CO 2D (Sigma‐Aldrich)
  • CH 3CD 2CO 2D (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‐CH 2CH=CH 2 (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

  • 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

  • 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

  • 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 H 2O (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

  • 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

  • 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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Literature Cited

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