RESUMEN
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.
Asunto(s)
Proteínas Sanguíneas/química , Análisis por Micromatrices/métodos , Animales , Anticuerpos/inmunología , Proteínas Sanguíneas/genética , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Conejos , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Anticuerpos/química , Análisis por Matrices de Proteínas , Proteoma/química , Animales , Anticuerpos/aislamiento & purificación , Especificidad de Anticuerpos , Cromatografía de Afinidad , Humanos , Inmunohistoquímica/métodos , Proteoma/inmunología , Proteoma/aislamiento & purificación , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
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, approximately 400,000 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.