Lyssna Antibody-Based Proteomics
Antibody-based proteomics provides the means for a systematic generation and use of antibodies to functionally explore the proteome. Such proteomics studies can be accomplished with the generation of “probes” in the form of protein-specific antibodies, but the lack of validated affinity reagents on a proteome-wide scale has up to now hampered such studies. The Swedish Human Protein Atlas (HPA) program, funded by the Knut and Alice Wallenberg Foundation, has been set up to allow for such a systematic exploration of the human proteome using Antibody-based proteomics. This is accomplished by combining high-throughput generation of affinity-purified (mono-specific) antibodies with protein profiling in a multitude of tissues/cell types assembled in tissue microarrays. The program is run by Proteome Resource (HPR) Center located at the School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm and here at Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Figure showing the different modules and the work-flow within the HPR project
As part of the main program, antibodies are used to explore protein expression patterns in a large variety of cells and tissues. As expression profiling is a main focus of the Uppsala group, we have organized the work into three sub-groups: (i) the immunohistochemistry group, (ii) the tissue microarrays group and (iii) the annotation group. The antibodies used in the study are derived from two sources; (i) mono-specific antibodies (affinity purified polyclonal antibodies) generated at the KTH site and (ii) antibodies provided by external antibody providers (commercial and academic sources). Protein profiling is based on immunohistochemistry applied on a multitude of tissues and cells in a tissue microarray (TMA) format. In the screening effort 9 different TMAs are used to analyze protein expression patterns in (i) 67 normal cell types from 48 different normal tissues, (ii) tumor cells from 216 individually different human cancers corresponding to the 20 most common forms of human cancer and (iii) 47 in vitro cultured cell lines and 12 patient cell samples. All immunostained TMAs are scanned to generate high-quality images. Images representing tissues are analyzed and annotated by pathologists or specially trained personnel and images representing cells are analyzed using and automated image analysis system. All images and annotations are freely available at the Human Protein Atlas portal (www.proteinatlas.org). In addition to generating a map of protein expression patterns, the Human Protein Atlas provides a database that can be used as a starting point to search for biomarkers.
Figure showing an example of a search for proteins with a high level of expression in kidney glomeruli and no expression in kidney tubules (a). In this example expression patterns (b) for the TDGF1 protein are displayed in both medium sized view (c) and in the high power view (d).