Human Protein Atlas (HPA) Project Workflow
Since 2003, the Human Protein Atlas (HPA) Project has been exploring the human proteome through antibody-based proteomics, which combines the high-throughput generation of antibodies with protein profiling in standard sets of tissue and cell microarrays. This exploration is achieved in three phases: 1) Antibody Generation, 2) Application Specific Validation, and 3) Protein Expression Profiling.
With the use of specially developed antigen design software, each antibody produced begins with the selection of a protein epitope signature tag (PrEST). The PrEST fragment is typically 50-150 amino acids in length and is as dissimilar as possible to other proteins. Special care is taken to avoid transmembrane regions, signal peptides, and high regional and/or sequence identity to other human proteins. The recombinant PrEST antigen is tested through a series of quality assurance steps. Plasmid inserts are sequenced and the size of the resulting recombinant protein is analyzed using mass spectrometry to ensure that the correct antigen has been produced and purified. The recombinant PrEST protein is then utilized as the antigen for immunization.
The antibodies undergo a three step affinity purification process: 1) Removal of tag-specific antibodies through depletion step, 2) Collection of antigen specific antibodies utilizing PrEST specific columns, and 3) Desalting and buffer exchange to obtain an optimal environment for long term antibody storage. To ensure antigen specificity, the purified antibodies are tested on protein arrays spotted with 384 randomly selected PrEST protein fragments.
Application Specific Validation
Every single antibody developed by the HPA is validated for use in immunohistochemistry (IHC). Each antibody is analyzed using:
- 48 normal human tissues in triplicate
- 20 of the most common cancer tissues
- 46 cell lines
- 12 clinical cell types
Over 700 IHC images are generated with each image annotated and curated by certified pathologists or by specially educated research scientists. In addition, the antibodies are tested by Western blot (WB) analysis and immunofluorescent (IF) microscopy.
Western blot analysis is performed using a standard sample setup composed of five different human protein lysates (cell lines RT-4 and U-251MG, plasma, liver and tonsil tissue), with a standardized protocol. In addition, certain antibodies are tested in WB using overexpressed lysates.
For immunofluorescent microscopy, each antibody has been analyzed in three human cell lines. The results are visualized with organelle probes displayed in different channels. As with IHC, all IF images are manually annotated with information on subcellular location, staining intensity, and staining characteristic.
Protein Expression Profiling
Once the antibody validation is complete, each application is assigned a validation score based on conformance with bioinformatic predictions and literature data. In addition, different antibodies against the same target are further annotated by comparing similarity in the different applications. Antibodies showing similar staining patterns strengthen the reliability of these antibodies even more. All of the images and data are available publicly on the Human Protein Atlas website www.proteinatlas.org.