Array-based Proteomics Gerald Walter, Biorchard AS, Oslo, Norway Konrad Bu Èssow, Max Planck Institute of Molecular Genetics, Berlin, Germany Zoltan Konthur, Max Planck Institute of Molecular Genetics, Berlin, Germany Angelika Lueking, Max Planck Institute of Molecular Genetics, Berlin, Germany Jo Èrn Glo Èkler, Max Planck Institute of Molecular Genetics, Berlin, Germany Ulrich Schneider, Max Planck Institute of Molecular Genetics, Berlin, Germany email: gerald@biorchard.com Array technology enables genome-wide screening for protein expression and interactions at high-throughput. Protein microarrays are emerging as a major technology platform for functional genomics. Protein Arrays as Technology Platforms for Functional Genomics 0727:1 Array technology enables genome-wide screening for protein expression and interactions at high-through- put Walter et al., 2000). The concept of the arrayed library and its display in high-density formats connects large numbers of de®ned clones to speci®c genes and protein products. Clone libraries become amenable to data integration including all steps from DNA sequencing to functional assays of gene products. The human genome gets annotated by building databases, interrelating genomic and proteomic data with clinical, population and environmental informa- tion. The collection and organization of biological material as libraries of clones requires automated, high-throughput technology. This technology allows the analysis of complex gene expression patterns, resulting in ®ngerprints of diseased versus normal or developmentally distinct tissues. cDNA expression libraries are displayed on high-density ®lters or microarrays and connect recombinant proteins to clones identi®ed by DNA hybridization or sequencing, hence creating a direct link between the gene catalog and a functional catalog Figure 1) BuÈssow et al., 1998, 2000). Following on from DNA microarrays and peptide spot synthesis Frank, 1992), fusion) proteins and antibodies Haab et al., 2000) or their expressing host cells de Wildt et al., 2000), are gridded at high density and used to study protein interactions in vitro and in vivo. Protein microarrays are emerging as a major technology platform for functional genomics. Protein Immobilization, Microfluidics and Detection 0727:2 For the construction of micro-) arrays or biochips, proteins are immobilized to solid surfaces either by noncovalent adsorption or covalent attachment. Recombinant proteins and mono- or polyclonal antibodies are noncovalently adsorbed onto hydro- phobic polyvinylidene di¯uoride PVDF) membranes BuÈssow et al., 1998) or poly-l lysine-coated micro- scope slides Haab et al., 2000). However, undirected adsorption may lead to the display of partially denatured proteins. Directed immobilization via tags or capture molecules, such as biotin-streptavidin, protein A or protein L de Wildt et al., 2000), can overcome this limitation and became a preferred strategy for immobilization of biomolecules onto glass/gold microarrays. Biosensor surfaces coated with charged, hydrophobic or chelating groups or biological capture molecules, such as antibodies, are now available e.g. Ciphergen's ProteinChip Array; Ciphergen Biosystems Inc, Fremont, CA, USA). For covalent attachment, glass surfaces are pretreated by coating with polyacrylamide or a dextran matrix, aminosilyation, silanization, speci®c linker attach- ments, N-hydroxysuccinimide NHS)-activation or polyethyleneglycol) polymer. Aldehyde slides Super- Aldehyde Substrates) are available from TeleChem International Cupertino, CA, USA) for easy access and compatibility with standard instrumentation. Alternatively, small molecules and proteins can be immobilized in an oriented fashion on chlorinated glass slides. The chlorine on the surface of the glass Array-based Proteomics ENCYCLOPEDIA OF THE HUMAN GENOME / &2003 Macmillan Publishers Ltd, Nature Publishing Group / www.ehgonline.net 1 Advanced Article contents  Protein Arrays as Technology Platforms for Functional Genomics  Protein Immobilization, Micro¯uidics and Detection  Recombinant Expression and Puri®cation for Entire Proteomes  Fusion Protein Arrays  Antibody Arrays  Peptide Aptamers, Mimotopes and Combinatorial Peptide Libraries  In vitro Protein-X Interactions on Microarrays  In vivo Protein Interactions: Yeast Two-hybrid System