ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 331 (2004) 303–313 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.05.008 Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions Birgit Kersten, ¤ Alexandra Possling, Franca Blaesing, Ekaterina Mirgorodskaya, Johan Gobom, and Harald Seitz Department Lehrach, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany Received 20 February 2004 Available online 17 June 2004 Abstract To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interac- tions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein–DNA inter- actions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate bind- ing site, the DnaA box. Interactions of DnaA domain 4 with a high-aYnity DnaA box (R4) and with a low-aYnity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433– 442 of Escherichia coli DnaA. This fragment contains six residues that were iden- tiWed as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein–DNA interactions.  2004 Elsevier Inc. All rights reserved. Keywords: Protein microarray technology; Fluorescence detection; NanoLC-MALDI-TOF MS; DnaA domain 4; DnaA box Protein–DNA interactions drive basic cellular pro- cesses such as transcription, replication, and recombina- tion. The identiWcation of such interactions and the determination of their speciWcity by measuring the eVects of mutations on the interaction are important to understand these processes. Direct methods, such as in vivo footprinting [1], allow target identiWcation of single DNA-binding proteins. A genome-wide location of DNA-binding proteins is enabled by the ChIP-chip approach, which combines chromatin immunoprecipitation with cDNA microarray hybridization and has recently been used to map transcriptional networks in yeast [2]. However, this method is limited by the need for antibodies against the DNA-binding proteins analyzed and does not easily allow including mutant DNA-binding proteins in the analysis. Several in vitro methods have been used for the anal- ysis of protein–DNA interactions. For a given DNA- binding protein, in vitro selection [3] has permitted the sampling of multiple DNA-binding sites. However, this method provides only a partial view of the binding site speciWcity because only the strongest interactions are selected. This limitation has been overcome by the devel- opment of dsDNA arrays for studying the DNA sequence speciWcity of protein–DNA interactions [4]. For given target DNA sites, phage display has emerged as a powerful tool, for example, for selecting transcription factors that recognize the given target out of millions of protein variants [5]. To study the eVects of individual protein mutants on the interaction, other methods that permit analysis of only one interaction at a ¤ Corresponding author. Fax: +49-30-8413 1128. E-mail address: birgit.kersten-berlin@gmx.de (B. Kersten).