Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ϳ400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.
A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.
There is a great need for comprehensive proteomic analysis of large patient cohorts of plasma and serum samples to identify biomarkers of human diseases. Here we describe a new antibody-based proteomic approach involving a reverse array format where serum samples are spotted on a microarray. This enables all samples to be screened for their content of a certain serum protein in a single experiment using target-recognizing antibodies and fluorescently labeled secondary antibodies. The procedure is illustrated with the analysis of the IgA levels in 2009 spotted serum samples, and the data are compared with clinical routine measurements. The results suggest that it is possible to simultaneously screen thousands of complex clinical serum samples for their content of the relative amount of specific serum proteins of clinical relevance. Molecular & Cellular Proteomics 4:1942-1947, 2005.The development of DNA microarrays has had an enormous impact in the research field of functional genomics where it has enabled large scale global analysis of whole genomes and transcriptomes. The developed methodology and technology have now become an established and relatively mature technique in terms of available instrumentation for production and analysis as well as commercially available premade microarrays, robust experimental protocols, and the long range of available tools and software for data analysis. Naturally there have been large efforts to continue the development of the microarray format with similar approaches for global protein analysis where great potential can be envisioned.The microarray technology makes it possible to analyze a large number of proteins within a small sample volume in a single experiment or with a reverse set-up analyze a limited number of proteins in many samples. The basic principles for highly sensitive "microspot" ligand binding assays were described by Ekins (1) and Ekins and Chu (2), who showed that small amounts of capture molecules in microspots can detect low concentrations of the analyte with high accuracy and sensitivity.The protein microarray applications can be divided into two categories: functional protein arrays and protein profiling arrays. Functional protein arrays can effectively screen large quantities of proteins for biochemical activity, protein-protein interactions, protein-lipid interactions, protein-nucleic acid interactions, and protein-small molecule interactions. The arrays have been used to study activity of uncharacterized proteins (3), antibody specificity profiling (4), and immune response profiling. Protein abundance arrays are used to measure protein abundance and/or alterations (5). Lately there has been progress in microarrays printed with antigen and used for detection of circulating antibodies in clinical specimens (6 -8). The protein profiling microarrays have also been used to measure the binding specificity of protein expression libraries (9, 10) and for protein profiling in cancer tissue (11).Currently there are two main types of profiling microarrays: forw...
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