Combinatorial Peptide Synthesis on a Microchip

Christopher Schirwitz1, Ines Block1, Kai König2, Alexander Nesterov2, Simon Fernandez1, Thomas Felgenhauer1, Klaus Leibe1, Gloria Torralba2, Michael Hausmann2, Volker Lindenstruth2, Volker Stadler1, Frank Breitling1, F. Ralf Bischoff1

1 German Cancer Research Center, Heidelberg, Germany, 2 University of Heidelberg, Kirchhoff Institute for Physics, Heidelberg, Germany
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.2
DOI:  10.1002/0471140864.ps1802s57
Online Posting Date:  August, 2009
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Abstract

Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom‐built microchips can be equipped with an amino‐modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer‐coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high‐complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm2 can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background‐free immunostaining on the chip‐based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high‐density peptide arrays. Curr. Protoc. Protein Sci. 57:18.2.1‐18.2.13. © 2009 by John Wiley & Sons, Inc.

Keywords: solid phase peptide synthesis; peptide array; combinatorial synthesis; atom transfer radical polymerization (ATRP); amino acid particles

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Combinatorial Peptide Synthesis on a Microchip
  • Support Protocol 1: Synthesis of the Atom Transfer Radical Polymerization (ATRP) Initiator
  • Support Protocol 2: Construction of Washing Chamber
  • Support Protocol 3: Construction of Coupling Chamber
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Combinatorial Peptide Synthesis on a Microchip

  Materials
  • 2‐bromo‐N‐(3‐triethoxysilyl)propyl isobutyramide (bromine silane; see protocol 2 for synthesis)
  • Dichloromethane (anhydrous, analytical grade)
  • Absolute ethanol (analytical grade)
  • Nitrogen (AlphaGaz 1N 2, AirLiquide, http://www.gaz‐industriels.airliquide.com/)
  • 2,2′‐bipyridyl (bipy, 98%; e.g., Sigma‐Aldrich)
  • Methanol (analytical grade)
  • Poly(ethyleneglycol) methacrylate (PEGMA, mol. wt., ∼360 g/mol; e.g., Sigma‐Aldrich)
  • Copper(I) bromide (analytical grade)
  • N,N‐dimethylformamide (DMF; anhydrous, dried over 0.4‐nm molecular sieve)
  • Fmoc‐β‐alanine (>99%; Iris BioTechnology; http://www.irisbiotech.com/)
  • N,N′‐diisopropyl carbodiimide (DIC, purum; e.g., Sigma‐Aldrich)
  • N‐methyl‐imidazole (NMI, analytical grade; e.g., Sigma‐Aldrich)
  • Acetic anhydride (analytical grade)
  • N,N‐diisopropylethylamine (DIPEA, analytical grade; e.g., Sigma‐Aldrich)
  • Piperidine (≥99%)
  • Amino acid particles (manufactured at German Cancer Research Center, Heidelberg; for further information and composition, see Beyer et al., ; Stadler et al., ; http://www.pepperprint.com)
  • Triisobutylsilane (TIBS; ultrapure; e.g., Sigma‐Aldrich)
  • Chloroform (analytical grade)
  • Trifluoroacetic acid (TFA, 99%)
  • 1× TBS‐T (see recipe)
  • Specific antibodies: mouse monoclonal anti‐FLAG M5 and rabbit anti‐HA (both from Sigma‐Aldrich)
  • Secondary antibodies: Alexa Fluor 647–conjuated goat anti‐rabbit IgG (H+L) and Alexa Fluor 546–conjugated goat anti‐mouse IgG (H+L): both available from Invitrogen
  • Microchips: designed at the Kirchhoff Institute for Physics, University of Heidelberg, and manufactured at the Institute for Microelectronics, Stuttgart (IMS‐CHIPS, http://www.ims‐chips.de)
  • Polypropylene tweezers (Rubis, model K35A; http://www.rubistweezers.com/)
  • 150‐W mercury vapor lamp (model TQ 150, Heraeus Noblelight)
  • Source of filtered, dried compressed air
  • 90° and 100°C ovens
  • Schlenk flask of appropriate size
  • Bath sonicator (e.g., VWR Ultrasonic Cleaner, model USC1200TH)
  • Fritted funnel with sintered glass disc, fine pore size
  • Vacuum desiccator
  • Glass petri dish
  • Rocking shaker: e.g., Duomax 2030 (Heidolph GmbH; http://www.heidolph‐instruments.de)
  • Plexiglas aerosol generators for triboelectric charging and amino acid microparticle deposition (custom‐built, Kirchhoff Institute for Physics, University of Heidelberg; for further information see Online Supporting Material in Beyer et al., )
  • Coupling chambers with two gas valves (see protocol 4)
  • Washing chambers and Teflon shield (see protocol 3)
  • Circuit boards and bonding wires (designed at Kirchhoff Institute for Physics, University of Heidelberg, http://www.kip.uni‐heidelberg.de; manufactured at Würth Elektronik GmbH & Co KG, Niedernhall, Germany)
  • Magnetic stirrer and magnetic stir bar (model RCT basic IKAMAG safety control, VWR International)
  • GenePix 4000B fluorescence scanner (Molecular Devices)
  • Chip holder adjusted to the standard scanning format (custom‐built metal plate in standard microscopy slide size; equipped with a chip‐size gap; tested at Molecular Devices, Munich, Germany)
  • GenePix Pro 4.0 Microarray Image Analysis software (Molecular Devices)
CAUTION: Ensure appropriate disposal of halogenated organic solvents. See appendix 2A for guidelines.NOTE: Unless otherwise noted, solely Milli‐Q‐filtered water (resistivity ∼18.2 MΩ cm) should be used.

Support Protocol 1: Synthesis of the Atom Transfer Radical Polymerization (ATRP) Initiator

  Materials
  • Triethylamine (99.5%)
  • 3‐aminopropyltriethoxysilane (99%; e.g., Sigma‐Aldrich)
  • Dichloromethane (anhydrous, analytical grade)
  • Nitrogen (AlphaGaz 1N 2, AirLiquide, http://www.gaz‐industriels.airliquide.com/)
  • 2‐bromoisobutyryl bromide (98%; e.g., Sigma‐Aldrich)
  • n‐hexane (analytical grade)
  • Dewar vessel containing dry ice/ethanol bath at –80°C
  • Schlenk flask of appropriate size
  • Magnetic stirrer and magnetic stir bar (model RCT basic IKAMAG safety control, VWR International)
  • Fritted funnel with sintered glass disc, fine pore size
  • Vacuum distillation apparatus
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Figures

Videos

Literature Cited

Literature Cited
   Beyer, M., Felgenhauer, T., Bischoff, F.R., Breitling, F., and Stadler, V. 2006. A novel glass‐slide based peptide array support with high functionality resisting non‐specific protein adsorption. Biomaterials 27:3505‐3514.
   Beyer, M., Nesterov, A., Block, I., König, K., Felgenhauer, T., Fernandez, S., Leibe, K., Torralba, G., Hausmann, M., Trunk, U., Lindenstruth, V., Bischoff, F.R., Stadler, V., and Breitling, F. 2007. Combinatorial synthesis of peptide arrays onto a microchip. Science 318:1888.
   Eichler, J. 2005. Synthetic peptide arrays and peptide combinatorial libraries for the exploration of protein‐ligand interactions and the design of protein inhibitors. Comb. Chem. High Throughput Screen. 8:135‐143.
   Fodor, S.P.A., Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T., and Solas, D. 1991. Light‐directed, spatially addressable parallel chemical synthesis. Science 251:767‐773.
   Frank, R. 1992. Spot synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48:9217‐9232.
   Frank, R. 2002a. High density peptide microarrays: Emerging tools for functional genomics and proteomics. Comb. Chem. High Throughput Screen. 5:429‐440.
   Frank, R. 2002b. The SPOT synthesis technique: Synthetic peptide arrays on membrane supports—principles and applications. J. Immunol. Methods 267:13‐26.
   Min, D.‐H. and Mrksich, M. 2004. Peptide arrays: Towards routine implementation. Curr. Opin. Chem. Biol. 8:554‐558.
   Pellois, J.P., Zhou, X., Srivannavit, O., Zhou, T., Gulari, E., and Gao, X. 2002. Individually addressable parallel peptide synthesis on a microchip. Nat. Biotechnol. 20:922‐926.
   Stadler, V., Beyer, M., König, K., Nesterov, A., Torralba, G., Lindenstruth, V., Hausmann, M., Bischoff, F.R., and Breitling, F. 2007. Multifunctional CMOS microchip coatings for protein and peptide arrays. J. Proteome Res. 6:3197‐3202.
   Stadler, V., Felgenhauer, T., Beyer, M., Fernandez, S., Leibe, K., Gröning, M., König, K., Torralba, G., Hausmann, M., Lindenstruth, V., Nesterov, A., Block, I., Pipkorn, R., Poustka, A., Bischoff, F.R., and Breitling, F. 2008. Combinatorial synthesis of peptide arrays with a laser printer. Angew Chem. Int. Ed. Engl. 47:7132‐7135.
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