Combinatorial Library Screening Coupled to Mass Spectrometry to Identify Valuable Cyclic Peptides

Silvia A. Camperi1, Silvana L. Giudicessi1, María C. Martínez‐Ceron1, Juan M. Gurevich‐Messina1, Soledad L. Saavedra2, Gerardo Acosta3, Osvaldo Cascone1, Rosa Erra‐Balsells4, Fernando Albericio5

1 National Scientific and Technological Research Council (CONICET), Buenos Aires, Argentina, 2 NANOBIOTEC Institute, UBA‐CONICET, Cathedra of Biotechnology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina, 3 CIBER‐BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Barcelona, Spain, 4 CIHDECAR‐CONICET, Department of Organic Chemistry, School of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires, Argentina, 5 School of Chemistry & Physics, University of Kwazulu‐Natal, Durban, South Africa
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.2
Online Posting Date:  June, 2016
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Abstract

Combinatorial library screening coupled to mass spectrometry (MS) analysis is a practical approach to identify useful peptides. Cyclic peptides can have high biological activity, selectivity, and affinity for target proteins, and high stability against proteolytic degradation. Here we describe two strategies to prepare combinatorial libraries suitable for MS analysis to accelerate the discovery of cyclic peptide structures. Both approaches use ChemMatrix resin and the linker 4‐hydroxymethylbenzoic acid. One strategy involves the synthesis of a one‐bead–two‐peptides library in which each bead contains both the cyclic peptide and its linear counterpart to facilitate MS analysis. The other protocol is based on the synthesis of a cyclic depsipeptide library in which a glycolamidic ester group is incorporated by adding glycolic acid. After library screening, the ring is opened and the peptide is released simultaneously for subsequent MS analysis. © 2016 by John Wiley & Sons, Inc.

Keywords: affinity; ChemMatrix; combinatorial; depsipeptides; MALDI MS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Cyclo‐(1,8)‐Ala‐X1X2X3X4X5‐Val‐Asp‐Gly‐HMBA‐CM Library with the Corresponding Linear Code Ala‐X1X2X3X4X5‐Val‐Ala‐Gly‐HMBA‐CM
  • Alternate Protocol 1: Synthesis of Cyclo‐(1,8)‐Ala‐OCH2CO‐X1X2X3X4X5‐Asp‐Ala‐Gly‐ HMBA‐CM Library
  • Support Protocol 1: Ninhydrin (Kaiser) Test
  • Support Protocol 2: Chloranil Test
  • Basic Protocol 2: Two‐Stage Combinatorial Cyclic‐Peptide Library Screening
  • Support Protocol 3: Target Protein Labeling with Texas Red or Biotin
  • Basic Protocol 3: MALDI‐TOF/TOF MS/MS Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Cyclo‐(1,8)‐Ala‐X1X2X3X4X5‐Val‐Asp‐Gly‐HMBA‐CM Library with the Corresponding Linear Code Ala‐X1X2X3X4X5‐Val‐Ala‐Gly‐HMBA‐CM

  Materials
  • Hydroxymethylbenzamide‐ChemMatrix resin (HMBA‐CM; PCAS BioMatrix Inc., cat. no. 4040)
  • Dichloromethane (DCM; Sigma‐Aldrich, cat. no. 270997)
  • N,N‐dimethylformamide (DMF; Sigma‐Aldrich, cat. no. 227056)
  • 4‐(N,N‐dimethylamino)pyridine (DMAP; Sigma‐Aldrich, cat. no. 522805)
  • Fluorenylmethoxycarbonyl (Fmoc) amino acids with side chain‐protecting groups (Peptides International Inc or Bachem Americas Inc)
  • N,N′‐Diisopropylcarbodiimide (DIPCDI; Sigma‐Aldrich, cat. no. 38370)
  • Acetic anhydride
  • 20% (v/v) piperidine in DMF (see recipe)
  • 1‐[bis(dimethylamino)methylene]‐1 H‐benzotriazolium 3‐oxide tetrafluoroborate (TBTU; Peptides International Inc., cat. no. KTB‐1066‐PI)
  • N,N‐diisopropylethylamine (DIPEA; Sigma‐Aldrich, cat. no. D125806)
  • OPp cleavage cocktail (see recipe), prepare just before use
  • (Benzotriazol‐1‐yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP; Sigma‐Aldrich, cat. no. 377848)
  • Hydroxy‐7‐azabenzotriazole (HOAt; Sigma‐Aldrich, cat. no. 41996)
  • 1‐[bis(dimethylamino)methylene]‐1 H‐benzotriazolium 3‐oxide hexafluorophosphate (HBTU; Sigma‐Aldrich, cat. no. 12804)
  • Protecting group cleavage cocktail (see recipe), prepare just before use
  • Acetonitrile
  • Acetic acid
  • Fume hood
  • Empty solid phase extraction cartridges, 1‐ to 60‐ml (Agilent Technologies, cat. no. 12131007 to 12131016).
  • Polypropylene frits for solid phase extraction cartridges, 20‐μm, 6.4‐ to 67‐mm (Agilent Technologies, cat. no. 12131019 to 12131024)
  • Manual pipette, 100‐ to 1,000‐μl (Eppendorf, cat. no. 492000904)
  • Pipette tips, 50‐ to 1,000‐μl, 71 mm (Eppendorf, cat. no. 0030 000.919)
  • Vacuum manifolds to evacuate the fluid from each syringe by filtration (e.g., Promega, Supelco).
  • Orbital shaker
NOTE: Perform synthesis inside a fume hood, and wear gloves, eye protection, and a lab coat. Follow all local, state, and federal safety regulations when disposing of chemical waste.

Alternate Protocol 1: Synthesis of Cyclo‐(1,8)‐Ala‐OCH2CO‐X1X2X3X4X5‐Asp‐Ala‐Gly‐ HMBA‐CM Library

  Additional Materials (also see protocol 1)
  • Glycolic acid (Sigma‐Aldrich, cat. no. 124737).
  • 1‐hydroxybenzotriazole (HOBt; Peptides International, cat. no. KHB‐1065‐PI).
  • N‐methyl‐2‐pyrrolidone (NMP; Sigma‐Aldrich, cat. no. 328634).
NOTE: Perform synthesis inside a fume hood, and wear gloves, eye protection, and a lab coat. Follow all local, state, and federal safety regulations when disposing of chemical waste.

Support Protocol 1: Ninhydrin (Kaiser) Test

  Materials
  • Peptidyl‐resin from protocol 1 or protocol 2
  • DCM
  • Solution A (see recipe)
  • Solution B (see recipe)
  • Drying oven

Support Protocol 2: Chloranil Test

  Materials
  • Peptidyl‐resin from protocol 1 or protocol 2
  • Acetone
  • Saturated chloranil solution in toluene: In a conical microcentrifuge tube mix 25 mg of chloranil and 1 ml of toluene. Centrifuge and recover the supernatant. Store protected from light at 2° to 8°C.

Basic Protocol 2: Two‐Stage Combinatorial Cyclic‐Peptide Library Screening

  Materials
  • Combinatorial cyclic‐peptide library (see protocol 1 and protocol 2)
  • DCM
  • DMF
  • Phosphate buffered saline (PBS; see recipe)
  • Skim milk
  • Bovine serum albumin, fraction V (BSA; Sigma‐Aldrich, cat. no. A8531).
  • Tween 20 (Sigma‐Aldrich, cat. no. P9416).
  • Target protein conjugated to Texas Red (see protocol 6).
  • Target protein conjugated to biotin (see protocol 6).
  • COPAS GP Sheath (Union Biometrica, cat. no. 300‐5070‐000).
  • Acetic acid
  • Acetonitrile
  • Streptavidin‐conjugated peroxidase (SA‐POD; Sigma‐Aldrich, cat. no. S5512)
  • 4‐chloro‐1‐naphthol (Sigma‐Aldrich, cat. no. C8890)
  • Methanol
  • 3,3′‐diaminobenzidine (DAB) tablets (Sigma‐Aldrich, cat. no. D4293)
  • 30% H 2O 2 (Sigma‐Aldrich, cat. no. 216763).
  • Solid‐phase extraction cartridge fitted with a polypropylene frit
  • COPAS BIOBEAD flow sorting equipment (Union Biometrica).
  • Leica MZ FLIII stereoscopic microscope (Leica Microsystems GmbH, Wetzlar, Germany).
  • Orbital Shaker

Support Protocol 3: Target Protein Labeling with Texas Red or Biotin

  Materials
  • N‐hydroxysuccinimide (NHS)‐Biotin (Sigma‐Aldrich, cat. no. H1759).
  • Texas Red sulfonyl chloride, 10 × 1 mg (Thermo Fisher Scientific, cat. no. T1905).
  • Labeling buffer (see recipe)
  • PD‐10 desalting column (5 kDa exclusion limit; GE Healthcare, cat. no. 17‐0851‐01).
  • Phosphate buffered saline (see recipe)
NOTE: It is highly recommended to read the instructions of the suppliers.NOTE: Perform steps 1 and 2 on ice.

Basic Protocol 3: MALDI‐TOF/TOF MS/MS Analysis

  Materials
  • Positive peptide‐beads (see protocol 7)
  • 30% (v/v) ammonium hydroxide
  • Acetic acid
  • Acetonitrile
  • Matrix solution (see recipe), prepared just before use
  • Drying chamber
  • Target plate
  • MALDI TOF/TOF MS
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Figures

Videos

Literature Cited

Literature Cited
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