SPOT synthesis: Reliability of array-based measurement of peptide binding affinity Armin A. Weiser a,1 , Michal Or-Guil b,1 , Victor Tapia b , Astrid Leichsenring c , Johannes Schuchhardt d , Cornelius Fro ¨ mmel a , Rudolf Volkmer-Engert c, * a Institute of Biochemistry, Charite ´, Universita ¨ tsmedizin Berlin, Monbijoustr. 2, 10117 Berlin, Germany b Institute of Theoretical Biology, Humboldt University, Invalidenstrasse 43, 10115 Berlin, Germany c Institute of Medical Immunology, Charite ´, Universita ¨ tsmedizin Berlin, Hessische Str. 3-4, 10115 Berlin, Germany d MicroDiscovery GmbH, Marienburger Str. 1, 10405 Berlin, Germany Received 23 February 2005 Available online 23 May 2005 Abstract Peptide arrays prepared by the SPOT synthesis technology have emerged as a proteomic tool to study molecular recognition and identify biologically active peptides. However, it was previously not clear how accurately signal intensities obtained by probing pep- tide arrays for protein binding really reflect the dissociation constants of the protein–peptide complexes. Using the monoclonal anti- body CB4-1 as a model system, we systematically compared dissociation constants of antibody–peptide complexes with signal intensities obtained using the SPOT technology. By analyzing a set of peptides possessing different affinities to the antibody, we determined the strengths of the SPOT screening method. The accuracy of the measured results was improved by taking regional trends in the membrane surface into account. A model based on the mass action law compares well with the experimental results. Interestingly, the applied concentrations of the binding partners do not directly correspond to the effective concentrations in the assay. We show that the SPOT technology is an accurate method for assigning the spotsÕ measured signal intensities to three different binding affinity classes. The dissociation constants of the intermediate region were found to be between pK dis = 5 and pK dis = 7. Altering the experimental parameters causes a directed change of this region. Ó 2005 Elsevier Inc. All rights reserved. Keywords: SPOT technology; Synthetic peptide arrays; Binding affinity; Dissociation constant; Effective concentration; Mass action law; Competition As demonstrated by DNA microarray experiments (‘‘DNA chips’’), the array format is a robust, reliable, and now well-established method for large-scale analysis of gene expression, allowing a global view of a living cell or organismÕs transcriptome based on a single experi- ment [1,2]. However, it became obvious that the infor- mation obtained from DNA chips is not sufficient for understanding life in general. Gene expression levels do not closely correlate with the abundance of func- tional protein molecules—the principal components in organizing and mediating cellular physiological func- tions via protein–protein networks [3]. One of the pri- mary goals in functional genomics is to understand how such networks support cell physiology. Ideally, we would like to draw a map displaying all protein inter- actions in the cell and then trace functional pathways along lines connecting the proteins. Recent interaction analyses produce an intricate picture and interpretation of the data is still inadequate [4–7]. We have to consider that the wiring between all components of the intricate network is dynamic. Protein complex formation is influ- enced by all the parameters that determine association 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.04.033 * Corresponding author. Fax: +49 30 450 524942. E-mail address: rve@charite.de (R. Volkmer-Engert). 1 These two authors have contributed equally. www.elsevier.com/locate/yabio Analytical Biochemistry 342 (2005) 300–311 ANALYTICAL BIOCHEMISTRY