Protein microarrays: catching the proteome Oliver Poetz, Jochen M. Schwenk, Stefan Kramer, Dieter Stoll, Markus F. Templin, Thomas O. Joos * NMI Natural and Medical Sciences Institute at the University of Tu ¨bingen, Markwiesenstr. 55, 72770 Reutlingen, Germany Available online 20 October 2004 Abstract After the completion of the human genome sequencing project, DNA microarrays and sophisticated bioinformatics platforms give scientists a global view of biological systems. In today’s proteome era, efforts are undertaken to adapt microarray technology in order to analyse the expression of a large number of proteins simultaneously and screen entire genomes for proteins that interact with particular factors, catalyse particular reactions, act as substrates for protein-modifying enzymes and/or as targets of autoimmune responses. In this review, we will summarise the current stage of protein microarray technology. We will focus on the latest fields of application for the simultaneous determination of a variety of parameters from a minute amount of sample. Future challenges of this cutting-edge technology will be discussed. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Protein microarray; Parallelised assay; Proteomics 1. Introduction Protein microarray technology has been successfully applied for the identification, quantification and functional analysis of proteins in basic and applied proteome research (MacBeath, 2002; Templin et al., 2003). These miniaturised and parallelised assay systems bear great potential as a replacement of singleplex analysis systems. The growing demands of genomics and proteomics for the analysis of gene and protein function in a global perspective has increased interest in microarray-based assays enormously. The basic principles of miniaturised and parallelised ligand binding assays were already described in the early 1980s by Roger Ekins’ ambient analyte theory (Ekins, 1989). Today, DNA microarrays are well-established high-throughput hybridisation systems that enable the exploration of the whole transcriptome in a single experiment (Anon., 2002). However, there is no absolute correlation between mRNA expression levels and corresponding protein expression (Gygi et al., 1999). Furthermore, it is impossible to deduce the functional state of a protein purely from its expression level. Therefore, additional high-throughput technologies are required that will facilitate the analysis of the function of the proteome. Within the last few years, microarray technology has expanded beyond DNA chips. A large variety of protein microarray-based approaches have already demonstrated that this technology is capable of filling the gap between genomics and proteomics. Depending on the field of application, protein micro- arrays can be classified into two categories: (1) Arrays for proteomics or focused protein profiling and (2) arrays for functional studies. The first category can be subdivided by array format into forward- and reverse-phase protein microarrays (Fig. 1). The difference between the two refers to the way the sample is applied. On forward-phase protein arrays, the sample is incubated on the array so that different analytes can be detected simultaneously. Examples include antibody microarrays that are used for the identification and quantification of target proteins. Reverse-phase arrays are the latest protein microarray developments. The array consists of different samples that are immobilised on a chip. In a single step, a large collection of probes can be screened for the presence or absence of one distinct target protein (Bouwman et al., 2003; Kononen et al., 1998; Paweletz et al., 2001; Petricoin and Liotta, 2002; Yan et al., 2003). www.elsevier.com/locate/mechagedev Mechanisms of Ageing and Development 126 (2005) 161–170 * Corresponding author. E-mail address: joos@nmi.de (T.O. Joos). 0047-6374/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2004.09.030