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OrganizationThe Human Protein Atlas is lead by the Executive Management Group, chaired by Mathias Uhlén, and receives strategic input and scientific advice regarding long-term goals, research priorities, and potential areas of expansion from the Steering board and an international Scientific Advisory Board. The resource is created and maintained by a large group of key staff. Steering BoardMathias Uhlen (chair), Royal Institute of Technology Scientific Advisory BoardJens Nielsen, Denmark (chair) - Systems biology AlbaNova site, StockholmAntigen and Antibody FactoryHead: Dr Hanna Tegel Responsibility: (i) Production of recombinant PrEST expression clones including cDNA synthesis, cloning, and plasmid purification. (ii) Production and purification of PrEST-proteins used for preparation of antigens and affinity columns. (iii) Management of immunization outsourcing. (iv) Generation of purified antibodies through affinity purification of polyclonal antisera. (v) Western blot (WB) analysis of antibodies approved in protein array analysis. (vi) WB antibody validation using over-expression lysates. (vii) Storage and distribution of antigens and antibodies within the program. (viii) Production of the Human Secretome in mammalian cells Description: Protein Epitope Signature Tags (PrESTs) are amplified with RT-PCR from total RNA template pools with specific oligonucleotide primers for each PrEST and then cloned into the expression vector. After transformation into E. coli the inserts are verified by DNA sequencing. The PrEST proteins are produced in shake flask cultures and then purified under denaturing conditions. The purified proteins that pass the quality control by SDS-PAGE and mass spectrometry are then used to prepare antigens used for immunization and affinity columns with PrEST-ligands. After immunization of the antigens the polyclonal antisera are carefully purified using the PrEST-ligand affinity columns and analyzed by WB. The group is also responsible for production of full-length proteins in mammalian cells within the Human Secretome project. Protein ScienceHead: Prof Sophia Hober Responsibility: Protein science research. Description: The module coordinates and conducts research projects aiming to extend the scientific outcome of data generated within the Human Protein Atlas project as well as improving current methodology. Both biological and technical research projects based on the vast amount of data generated are performed to further explore the function, localization and interactions of human proteins. SciLifeLab site, StockholmProtein Array TechnologiesHead: Prof Peter Nilsson Responsibility: To validate the specificity and selectivity of all purified HPA antibodies. To develop and utilize peptide, antigen and antibody based microarray methodologies for large scale analysis of body fluids in the context of biomarker discovery and autoantibody profiling. Description: Methodology for microarray based analysis of antibody specificity has been developed, where all purified antibodies are analyzed on protein arrays with immobilized PrESTs. Each microarray is divided into 21 replicated subarrays with 384 PrESTs, enabling the analysis of 21 antibodies simultaneously. The antibodies are detected through a fluorescently labeled secondary antibody. A specificity plot is generated for each antibody, where the signal from the binding to its antigen is compared to the unspecific binding to all the other PrESTs. A dual color system is used in order to verify the presence of the spotted PrESTs. Several complementary microarray formats for systematic analysis of body fluids are being utilized and under constant development. The PrEST-arrays have been implemented for systematic antigen-based plasma profiling for the screening of new autoimmunity components. The antibody microarrays with the possibility for simultaneous analysis of large amounts of analytes with high sensitivity and the reverse phase serum microarrays which enable serum from very large patient cohorts to be analyzed simultaneously are both utilizing in-house produced planar microarrays. The main platform for systematic antibody-based plasma profiling, is although the suspension bead array format with capacity for multiplexing in two dimensions, enabling the simultaneous profiling of 384 antibodies on 384 samples, see Plasma profiling. Subcellular ProfilingHead: Prof Emma Lundberg (Stanford) Team leader: Dr Ulrika Axelsson Responsibility: Creation of the subcellular section: (i) determination of the subcellular distribution of proteins using high-resolution confocal microscopy, (ii) validation of antibody specificity using gene editing and silencing technologies (iii) annotation and knowledge-based curation of subcellular distribution profiles. Description: The Subcellular Profiling group is responsible for determination of the spatiotemporal subcellular distribution of proteins in human cells. The distribution is systematically assessed at single-cell level using the antibodies generated within the Human Protein Atlas program. Each protein is studied in up to three cell lines, which are selected based on RNA expression levels from a panel of cell lines of different origin. Subcellular distribution is determined by indirect immunofluorescence followed by confocal microscopy. The resulting high-resolution images show the protein of interest as well as markers for the nucleus, microtubule cytoskeleton, and endoplasmic reticulum. This enables manual annotation of protein localization to one or more subcellular structures, as well as detection of cell-to-cell variability. The high-resolution confocal images, annotations, and gene expression data can be explored in an interactive gene-centric manner on the Subcellular section. The Subcellular Profiling group group uses different strategies for enhanced antibody validation. Most common is validation by independent antibodies, but we also perform genetic validation by knocking down the gene of interest using siRNA, and validation by comparative immunostaining in cells co-expressing a GFP-tagged recombinant version of the protein of interest. Plasma ProfilingHead: Prof Jochen M. Schwenk Responsibility: Utilize HPA antibodies for multiplexed immunoassays to profile circulating proteins. Description: The Plasma Profiling group is responsible for developing and applying antibody-based assays for protein biomarker analysis in human body fluids. The group has established a variety of high-throughput assays, where antibodies are immobilized to create affinity-based assays for soluble targets. For discovery, bead arrays are composed of up to 384 antibodies to analyze up to 384 samples at a time (direct labeling assays). For quantification and the analysis of protein interactions, the group developed a multiplexed workflow to select antibody pairs to capture and detect proteins via two distinct epitopes (sandwich assays). The Plasma Profiling group also established different procedures for validating the selectivity of antibodies, including paired antibodies, sequential affinity capture, or coupling the readout of protein capture to mass spectrometry. In addition, the group uses orthogonal methods, such as targeted mass spectrometry, other multiplexed immunoassay platforms. By integration of various data types, the group determines the association of target proteins with health and disease phenotypes. Bioinformatics and integrative omicsHead: Dr Linn Fagerberg Responsibility: Analysis and integration of large-scale biological data using advanced bioinformatic methods focusing on: (i) transcriptomics sequencing of cells and tissues; (ii) the quantitative transcriptomics-based classification of the human proteome; and (iii) advancing personalized medicine to allow for the profiling of human health and disease based on multi-level omics strategies. Description: The bioinformatics and integrative omics group is specialized in utilizing various methods for mining “big data”. The main focus is analyzing the human proteome and transcriptome using integrative approaches to perform a functional and spatial classification. The group is also leading the integrative data analysis within the Human Disease Blood Atlas effort, in which the omics-based profiling is expanded across a wide range of diseases based on well-characterized cohorts with the aim to characterize molecular fingerprints and allow stratification of patients to pave the way for future precision medicine efforts. Systems medicineHead: Assoc Prof Adil Mardinoglu Responsibility: Investigation of gene expressions in different cancers and cancer cell lines and their association with patient survival outcomes. Presentation of these results in the Pathology and Cell Line sections of the Human Protein Atlas. The group is also using systems biology tools to study complex human diseases. Description: The group's main focus is on examining gene expressions within a wide array of cancers and their corresponding cell lines. By linking these gene expressions with patient survival rates, the group hopes to shed light on possible prognostic markers and therapeutic targets. The results of their meticulous research are prominently displayed in the Human Protein Atlas, contributing to the wider scientific community's understanding of cancer pathology. Moreover, the group uses advanced systems biology tools to investigate complex human diseases. By constructing and analyzing biological networks, the aims to unravel the interconnected web of molecular interactions that underpin these conditions. This systems-level approach facilitates the identification of key molecular players and offers fresh insights into disease mechanisms, opening new avenues for diagnosis, prevention, and treatment strategies. . Single cell profilingHead: Dr Cheng Zhang Responsibility: The group specializes in utilizing single-cell omics data and bioinformatics tools to analyze gene expression and identify cell type-specific genes. Additionally, the group benchmarks gene expression in healthy cells to distinguish key cell types and gene targets implicated in diseases. Description: The group excels in the analysis and interpretation of single-cell omics data derived from human tissues. The primary focus is to meticulously profile the diverse array of human cell types and categorize genes based on their specificity to certain cell types. Further, the group harnesses the power of systems biology tools, such as multi-omics networks and genome-scale metabolic models, to integrate and interpret single-cell omics data. This approach is crucial for unraveling the molecular mechanisms underlying complex diseases that may evolve through interactions among multiple cell types. Additionally, the group aims to identify central pathways and hub genes that have the potential to serve as innovative biomarkers or therapeutic targets, tailoring the diagnosis and treatment to the individual needs of patients. Tissue cell type profilingHead: Dr Lynn Butler Responsibility: The development and application of computational-based analysis methods to profile cell type specificity of gene expression, within different tissue types. This data is presented in the Tissue Cell Type section of the Human Protein Atlas. Description: The group use correlation based integrative co-expression analysis to mine information on gene expression at the cell type level, using unfractionated bulk RNAseq data. We apply our method to existing large datasets, sourced though resources such as the GTEx consortium. Targeted Proteomics and Plasma ProfilingHead: Dr Fredrik Edfors Responsibility: The group is using mass spectrometry to validate reagents generated within the scope of the Human Protein Atlas. This includes orthogonal antibody validation by Capture MS, as well as bottom-up proteomics analysis of samples of human origin. Description: The group is focusing on the use of Stable Isotope-labeled Standard (SIS) Protein Fragments based on the PrEST sequences to precisely quantify and accurately quantify proteins across human tissues, cell-lines and in blood plasma. A wide variety of bottom-up proteomics techniques are used to generate targeted assays, including Selective Reaction Monitoring (SRM) and Data Independent Acquisition (DIA) strategies. IT, LIMS and data handlingHead: Kalle von Feilitzen Responsibility: (i) To deliver custom made software solutions for all operations in the Human Protein Atlas project, (ii) to provide the collected data to the public via the Human Protein Atlas, (iii) to map and quantify RNA-seq data, and (iv) to initiate the analysis of human proteins by in silico selection and design of Protein Epitope Signature Tags (PrESTs). Description: With the LIMS (Laboratory Information Management System) as the backbone, data is collected from each module in the pipeline. The protein expression profiles, RNA-seq data and raw data from the project is published on the Human Protein Atlas public web site through annual releases. The group is also involved in research performed in the project, such as data collection, data interpretation and visualization as well as statistical analysis. Web and social mediaHead: Dr. Åsa Sivertsson Responsibility: To be editor for the open access Human Protein Atlas resource. Responsible for section descriptions and general information about the resource. Also responsible for the News section, press releases and social media. Description: The group is responsible for the web, news, press releases and social media. Biomedicum site, Karolinska Institutet, StockholmBrain ProfilingHead: Dr Jan Mulder Advisor: Prof Tomas Hökfelt Responsibility: Generation of transcriptomics and antibody-based protein data for the Brain Atlas: (i) Validation of antibodies against human targets on rodent tissues, (ii) profiling the distribution of proteins in the developing, adult, and diseased nervous system, (iii) quantification, annotation, and presentation of whole brain protein distribution profiles, (iv) dissection and collection of brain samples (various species), (v) RNAsequencing of human brain and rodent tissues using the MGI DNBSEQ-T7 platform, (vi) 3D volume imaging of human and animal samples (iDISCO protocol). Description: The brain is a complex organ from a functional and anatomical perspective. To capture this complexity, we complemented our standard human tissue and organ analysis with a more in-depth analysis of the central nervous system by including more brain regions and by investigating regional protein expression in different mammalian species. By visualizing protein distribution in the ‘small’ mouse brain we are able to provide a more complete overview of protein distribution in the mammalian brain including most brain regions and cell types. For this, HPA antibodies against proteins expressed in the mouse nervous system are validated on mouse brain tissue using immunofluorescence and results are compared to available expression data. Antibodies that pass validation are used to generate detailed protein distribution profiles using 20-30 coronal sections of the mouse brain with a 400 μm section interval covering all major brain nuclei. Whole slide immunofluorescence captured at 10x primary objective is analyzed and regional, cellular and subcellular protein distributions are quantified. Data and images are optimized for online publication. Pig brain project: The pig brain transcriptomics project is a collaborative project between the Human Protein Atlas and the Lars Bolund institute of regenerative Medicine (Dr Yonglun Luo), BGI-Qingdao, China. Human prefrontal cortex project: The human prefrontal cortex project is a collaboration with Prof Miklós Palkovits, Human Brain Tissue Bank Microdissection Laboratory of the Semmelweis University, Budapest, Hungary Uppsala siteTissue ProfilingHead: Dr Cecilia Lindskog Advisor: Prof Fredrik Ponten Generation of antibody-based protein data for the Tissue and Pathology sections: (i) handling and processing of tissues (biobank material), (ii) handling and storage of antisera, (iii) testing of antibodies, (iv) immunohistochemical staining based on conventional or multiplex detection, (v) scanning of stained tissue slides and image processing, (vi) validation of antibody target specificity, (vii) annotation and final approval of immunohistochemically stained tissues, (viii) determination of antibody reliability based on enhanced antibody validation strategies, (ix) generation of knowledge-based protein expression profiles, and (x) prepare the data for release on the Tissue and Pathology sections. Tissue data curation: (i) restaining with novel antibodies or staining protocol for poorly characterized proteins, (ii) addition of extended tissue samples to cover rare cell types or temporal expression, (iii) in-depth annotation of existing tissue images to add data for more cell types Analysis of scRNA-seq based data for the Single cell type section: annotation and curation of single cell type clusters generated by the Single cell profiling group led by Dr. Cheng Zhang. Clinical pathology: (i) develop strategies to identify potential biomarkers based on the HPA database and other efforts, (ii) validate proteins that can be used as clinical biomarkers for disease, (iii) participate in clinical studies, collect tumor material and clinical data to generate specific cancer TMAs coupled to clinical databases, (iv) perform statistical analysis and validate the clinical usefulness of identified biomarkers. For a detailed description of the various methods used at the Uppsala site please visit the method summary pages of the Tissue, and Single cell type sections. |
The Human Protein Atlas project is funded
