Human Proteome Project? Màster en bioquímica, biologia molecular i biomedicina

1. Human Proteome Project? Màster en bioquímica, biologia molecular i biomedicina Mòdul 4: Genòmica i Proteòmica Núria Colomé Calls

2. After the Human Genome Project, the Human Proteome Project seems a natural succesor John Bergeron president of the Human Proteome Organisation (HUPO) and a group of leading researchers propose a large-scale assault on the human proteome.

3. This group plans to consult with the proteomics community for the first time at HUPO’s Amsterdam World Congress in August. Human Proteome Project is now feasible because: -The number of protein coding genes is around 21000 making the scale of human proteomics more manageable

4. Human Proteome Project would reveal: Which proteins are present in each tissue Where is located each protein in the cell Protein-protein interactions Invaluable information with clinical utility Difficulties: There are around 200 types of human cells that manufacture proteins at differing levels and different moments in time. Protein-coding genes in the body can make tens of different versions of a protein. Each of these can be modified by addition of chemical groups.

5. The researchers’ plan is to use different experimental approaches: Mass spectrometry : To identify proteins and their quantities Generate antibodies to each protein: to show its location in tissues and cells Systematic Identification protein-protein interactions Bioinformatics tools design: to pool and make accessible the data

6. Proteins Identification Initial assay: HUPO Human Plasma Proteome Project (HPPP)

7. Abundance and dynamic range of plasma proteins Proteins Identification Initial assay: HUPO Human Plasma Proteome Project (HPPP) 2002 the Plasma Proteome Project was initiated. The aim is the systematic characterization of circulating proteins in health and disease to facilitate development of biomarkers for prevention, diagnosis and therapy of different diseases There were 35 collaborating laboratories and multiple analytical groups that were provided with 1.0mL of serum and plasma by three different methods of anticoagulation for plasma. Different strategies to analyse the samples were used: - Depletion or not of abundant proteins: albumin and/or IgG and Top 6 - Fractionation of intact proteins: 2DE, LC or IEF methods - Peptides separation: Cation exchange and/or Reverse phase - Multiple mass spectrometry instruments: ESI MS/MS, MALDI MS and FT-ICR-MS - Varied search algorithms and databases: PepMiner, SEQUEST, MASCOT and VIPER

8. Proteins Identification Initial assay: HUPO Human Plasma Proteome Project (HPPP) Results 18 laboratories submitted a total of 42306 protein identifications. This initial assay has provided a core data set of 3020 proteins based on two or more peptide, without redundant entries and one representative protein among multiple proteins (homologs and isoforms) The laboratories that reported more results had used a depletion method, peptide chromatographic separation and MSMS instruments

9. Proteins Identification Initial assay: HUPO Human Plasma Proteome Project (HPPP) Comments There is a high variability on the results depending on sample preparation, fractionation and detection methods. Probably complementary. These results lay a foundation for development and validation of circulating protein biomarkers in health and disease and achieve quantitative comparisons across specimens, not just compositional analyses There is a Human Proteome contribution to human genome: 185 proteins are not associated with genes EnsEMBL and 118 peptides were identified as highly probable matches to ORFs in the human genome not previously known to have protein products

10. Generate antibodies to each protein to show its location Protein Atlas: Part of the HUPO Human Antibody Initiative (HAI)

11. Generate antibodies to each protein to show its location Protein Atlas: Part of the HUPO Human Antibody Initiative (HAI) The human protein atlas shows expression and localization of proteins in a large variety of normal human tissues, cancer tissues and cell lines with the aid of immunohistochemistry (IHC) images. Subcellular localization results for 769 antibodies in three different cells lines Atlas content: 3014 antibodies and 2,940,744 images.

12. Generate antibodies to each protein to show its location Protein Atlas: Part of the HUPO Human Antibody Initiative (HAI) HER2 Search with links to Uniprot, NCBI and EnsEMBL databases and antibody results

13. Generate antibodies to each protein to show its location Protein Atlas: Part of the HUPO Human Antibody Initiative (HAI) ERBB2 expression profiles for HPA001383 antibody in Normal tissues, Cancer tissues and Cell lines

14. Generate antibodies to each protein to show its location Protein Atlas: Part of the HUPO Human Antibody Initiative (HAI) ERBB2 expression profiles images for HPA001383 antibody in Breast Cancer Tissue

15. Generate antibodies to each protein to show its location Protein Atlas: Part of the HUPO Human Antibody Initiative (HAI) Cell Location of BCAN protein: Immunofluorescent (IF) images generated with confocal microscopy

16. Identification of Protein-protein interactions Large-scale mapping of human protein–protein interactions by mass spectrometry

17. Bait to bait conectivity map of bait proteins Focused view of selected bait-bait networks Identification of Protein-protein interactions Large-scale mapping of human protein–protein interactions by mass spectrometry This study presents the first large-scale study of protein–protein interactions in human cells using a Immunoprecipitation high-throughput mass spectrometry (IP-HTMS). The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. 6463 interactions were found The data set provides for both recapitulation of known complexes and discovery of new interactions and complexes.

18. Bioinformatics

19. Bioinformatics Develop a unique reservoire to pool and make accessible all the data generated since now Exist different datasets for human plasma or serum Healthy Human Individual’s Integrated Plasma proteome (HIP2) This database has been developed to be the comprehensive collection of healthy human plasma proteins, it contains mappings of supporting peptide evidence from several high quality and high-throughput mass spectrometry (MS) experimental datasets. Users can search for plasma protein/peptides annotations, peptide/protein alignments, and experimental/sample conditions.

20. Human Protein Atlas Bioinformatics Other repositories: PRIDE: PRoteomics IDEntifications database is a centralized, standards compliant, public data repository for proteomics data. PRIDE is able to capture details of post-translational modifications. This database contain: 3,745 Experiments,726,086 Identified Proteins , 4,618,998 Identified Peptides, 466,837 Unique Peptides and 6,446,100 Spectra Peptide Atlas: PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments Protein Atlas: The human protein atlas shows expression and localization of proteins

21. Opinions “The whole effort could take a decade and would involve more labs than the human genome project”. Bergeron, Nature News “Rather than analyse the proteome of one chromosome, it may be better to tackle the proteome of mitochondria or the cell membrane because it would reveal more about biology and diseases”. Steven Carr, Nature News “Proteome Project would involve many labs, with inevitable issues about data-sharing and competition”. Michael Washburn, Nature News “The project may have a better chance of success with stronger focus on diseases such as cancer, you need to show clinical utility of the approach before launching a mega-project”. Sudhir Srivastava, Nature News

22. References Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Gilbert S. Omenn et al., Proteomics 2005, 5, 3226–3245 Data management and preliminary data analysis in the pilot phase of the HUPO Plasma Proteome Project. Marcin Adamski et al., Proteomics 2005, 5, 3246–3261 Large-scale mapping of human protein–protein interactions by mass spectrometry. Rob M Ewing, Molecular Systems Biology 2007,3:89, 1-17 HIP2: An Online Database of Human Plasma Proteins from Healthy Individuals. Sudipto Saha,BMC Medical Genomics 2008, 1:12 http://www.nature.com/news/index.html http://www.proteinatlas.org/ http://www.hupo.org/ http://bio.informatics.iupui.edu/HIP2/ http://www.ebi.ac.uk/pride/init.do