ORGANIZATION

The Executive Management is responsible for the overall strategies of the consortium and for managing the overall program:

Prof Mathias Uhlen, Program Director
Dr Tove Alm, Program Coordinator, Head of Communications
Assoc prof Emma Lundberg, Director of Cell Atlas
Dr Cecilia Lindskog Bergström, Director of Tissue Atlas
Prof Fredrik Ponten, Director of Cancer Atlas
Dr Hanna Tegel, Director of protein and antibody production
Prof Peter Nilsson - Head of biomarker discovery and protein arrays
Prof Sophia Hober - Head of protein science
Kalle von Feilitzen, Director of Bioinformatics and IT
Dr Åsa Sivertsson, Head of Curation

AlbaNova site, Stockholm

SciLifeLab site, Stockholm

Uppsala site



AlbaNova site, Stockholm

Site Director:Dr Hanna Tegel

ModuleGroup leader
Antigen and Antibody FactoryDr Hanna Tegel
Epitope Mapping and Therapeutic Antibodies Dr Johan Rockberg
Protein ScienceProf Sophia Hober


Antigen and Antibody Factory

Group leader:

Dr Hanna Tegel

Personnel:

Delaram Afshari (research engineer), Roxana Astefanei (laboratory engineer), Anna Berling (team leader), Lucas Bremer (laboratory engineer), Carl-Fredrik Bowin (laboratory engineer), Annelie Cajander (research engineer), Tove Canerstam (laboratory engineer), Melanie Dannemeyer (researcher), Malin Ebbinge (laboratory engineer), Siri Ekblad (research engineer), Henric Enstedt (research engineer), Lili Gong (research engineer), Anneli Halldin (research engineer), Sara Kanje (researcher), Muna Muse (laboratory engineer), Anne-Sophie Svensson (team leader), Lan Lan Xu (laboratory engineer)

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.

Description:

PrEST regions are first amplified with RT-PCR from a total RNA template pool with the specific oligonucleotide primers designed in the Bioinformatics module. Three different RNA pools are used, two consisting of total RNA from six individual human tissues, and one comprising total RNA from 10 different cell lines. For PrESTs not found in any RNA pool, cDNA clones are used as template (Ultimate™ ORF LITE Clones (Human collection), Life Technologies, Carlsbad, CA and Mammalian Gene Collection (The MGC Project team, Genome Res. 2009)). Amplicons are automatically processed with solid phase restriction, and ligated into the plasmid vector pAff8c (Larsson, M. et al, 2000) where the human gene fragment is fused to a histidine tag and albumin binding protein (His6ABP). After transformation into E. coli Rosetta(DE3), (His6ABP). After transformation into E. coli Rosetta(DE3), inserts are verified by DNA sequencing to omit clones with mutations and approved clones are single cell streaked. Plasmids are collected from all purified clones for deposition in the clone library and glycerol stocks are prepared and used as starting material for protein production. All proteins are expressed as His6ABP fusions in E. coli shake flask cultures upon induction with IPTG. A fully automated protein purification system has been developed to allow for purifications of up to 60 cell lysates at a time. One-step purification is enabled by the hexahistidine affinity tag and metal affinity chromatography (IMAC) and performed under denaturing conditions. After evaluation of protein concentration and purity, the molecular weight of the PrEST proteins is determined by mass spectrometry as a final quality control. The purified proteins are then used to prepare antigens and affinity columns with PrEST-ligands. In addition, affinity resin with His6ABP-ligand is also produced.

The group is also responsible for the generation, purification and Western Blot analysis of all antibodies produced in the Human Protein Atlas (HPA) program. Polyclonal antisera generated together with collaborative partners are carefully purified in a three-step fashion consisting of: depletion of unwanted specificity, capture of wanted specificity and a final buffer exchange step. A manual process using gravity-flow columns carries out depletion of antibodies with unwanted specificity. The following steps are performed on the ÄKTAxpress chromatography system enabling a high-throughput semi-automated process where captured antibodies are eluted by a low pH glycine buffer and automatically loaded onto a desalting column for buffer exchange. Antibodies are supplemented with 50&37#; glycerol and 0.02&37#; sodium azide for long-term storage at -20°C. The binding specificity of all antibodies is determined on protein microarrays to certify that only antibodies with high specificity and low background binding are approved for immunohistochemistry analysis. All approved antibodies are further analyzed in a high-throughput WB platform using protein lysates from human cell lines (RT-4 and U-251 MG), human plasma depleted of IgG and HSA and whole tissue lysates from human liver and tonsil. Published antibodies scored as non-supportive, in the standard WB panel, are subsequently revalidated in a WB set-up comprising an over-expression lysate (VERIFY Tagged Antigen™, OriGene Technologies, Rockville, MD) as a positive control.

ABP - Albumin Binding Protein
IPTG - Isopropyl-B-D-Thiogalactopyranoside
IMAC - Immobilized Metal Affinity Chromatography

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Epitope Mapping and Therapeutic Antibodies

Group leader:

Asstn. Prof. Johan Rockberg

Personnel:

Francis Jingxin Hu (PhD-student), Magnus Lundqvist (PhD-student), Anna-Luisa Volk (PhD-student), Niklas Thalen (PhD-student), Mona Moradi (PhD-student)

Responsibility:

(i) To determine antibody epitopes for HPA antibodies, and(ii) to generate monoclonal reagents towards selected targets and screen them in functional assays for therapeutic effect.

Description:

Epitope mapping is performed on a selection of the HPA's monospecific and monoclonal reagents using a combination of platforms including: (i) cellsurface display of target directed peptide libraries using staphylococcal display, (ii) suspension bead arrays for mapping using overlapped or alanine mutated synthetic peptides, and (iii) large scale synthetic peptide arrays.

Monoclonal antibodies to clinically relevant target proteins are selected using a combination of phage display and cell surface display of antibody fragments. These are subsequently screened for desired effect in cell-based assays and cloned for production in mammalian cells as full-length antibodies.

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Protein Science

Group leader:

Prof Sophia Hober

Personnel:

Sara Kanje (researcher), Sarah Lindbo (PhD-student), Emma von Witting (PhD-student), Julia Scheffel (MSc-student)

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.

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SciLifeLab site, Stockholm

Site Director: Prof Peter Nilsson

ModuleGroup leader
Protein Array TechnologiesProf Peter Nilsson
Cell ProfilingAssoc prof Emma Lundberg
Plasma ProfilingAssoc prof Jochen M. Schwenk
Brain ProfilingDr Jan Mulder
BioinformaticsKalle von Feilitzen


Protein Array Technologies

Group leader:

Prof Peter Nilsson

Personnel:

Eni Andersson (research engineer), Burcu Ayoglu (researcher), Cecilia Hellström (research engineer), Anna Häggmark (researcher), David Just (PhD-student), Cecilia Mattsson (research engineer), Maria Mikus (PhD-student), Maja Neiman (researcher), Ulrika Qundos (researcher), Julia Remnestål (research engineer), Ronald Sjöberg (researcher), Arash Zandian (PhD-student)

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 bodyfluids 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.

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Cell Profiling

Group leader:

Assoc prof Emma Lundberg

Personnel:

Martin Hjelmare (lab manager), Dr. Lars Björk (lab manager), Hammou Ait Blal (research engineer), Anna Bäckström (research engineer), Frida Danielsson (PhD-student), Jenny Fall (technician), Christian Gnann (research engineer), Diana Mahdessian (PhD-student), Rutger Schutten (research engineer), Marie Skogs (PhD-student), Dr. Charlotte Stadler (researcher), Dr. Devin Sullivan (post-doc), Dr. Peter Thul (post-doc), Mikaela Wiking (PhD-student), Lovisa Åkesson (PhD-student)

Responsibility:

(i) Determine the subcellular distribution of proteins using high-resolution confocal microscopy, (ii) validation of antibody specificity using siRNA mediated gene silencing, (iii) validation of antibody specificity by co-staining of cells expressing GFP-tagged proteins and (iv) annotation and knowledge- based curation of subcellular distribution profiles.

Description:

The Cell Profiling module is responsible for the subcellular localization of proteins using antibodies generated in the Human Protein Atlas (HPA) program.

For each protein the subcellular localization is studied in three different human cell lines selected, based on RNA expression levels, from a panel of eighteen different cell lines. In order to localize the whole human proteome in one specific cell line, U-2 OS cells have been selected and are always used. The cell lines are cultured in vitro and immunofluorescently stained using automated protocols before high-resolution confocal images are acquired. All images are manually annotated and for each protein the subcellular localization, characteristics and intensity of the staining is described. To validate the results, the corresponding target protein may be down regulated by siRNA prior to immunostaining. Subsequently the loss of antibody staining is quantified and antibody specificity assessed. Alternatively, the results are validated by co-staining of cells expressing GFP-tagged proteins.

In the end, a knowledge-based curation of the subcellular distribution is performed in a gene-centric manner, taking into account the staining of one or multiple antibodies. The subcellular distribution is classified as main location and additional locations.

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Plasma Profiling

Group leader:

Assoc prof Jochen M. Schwenk

Personnel:

Eni Andersson (research engineer), Annika Bendes, (research engineer), Sofia Bergstršm (research engineer), Sanna Bystršm (PhD-student), Tea Dodig-Crnkovic (PhD-student), Kimi Drobin (PhD-student), Dr Claudia Fredolini (researcher), Dr Mun-Gwan Hong (biostatistician), Dr MariaJesus Iglesias (researcher), Dr Maja Neiman (researcher)

Responsibility:

Utilizing HPA antibodies in multiplexed affinity arrays to profile proteins in human serum and plasma for the discovery, identification and verification potential biomarker candidates.

Description:

Antibodies are immobilized on color-coded beads to create antibody arrays in suspension. Each bead array is composed of 384 antibodies that are selected either from defined lists of proteins (targeted design) or by antibody availability (random design). The samples are biotinylated and heat treated for the antibody array analysis, where protein profiles are generated for up to 384 samples at a time.

We are also expanding the profiling procedure into other body fluids (e.g. CSF, BAL or dried blood spots) and are conducting studies such as within different disease areas such as cancer, diabetes, cardiovascular, or neurodegenerative disorders. The data from such multiplexed single-binder assays is accordingly processed and analyzed with biostatistical models to identify which antibodies provide disease indications. Such antibodies then enter further validation assays using mass spectrometry and to develop dual-binder sandwich immunoassays. Subsequent studies are then designed to validate the candidate proteins in larger sample sets from preferably independent collection sites.

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Brain Profiling

Group leader:

Dr Jan Mulder

Personnel:

Agnieszka Limiszewska (technician), Dr. Nicholas Mitsios (researcher), Dr. Jaekyung Shin (researcher), Evelina Sjšstedt (PhD student), Sania Kheder (technician).

Responsibility:

(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.

Description:

Mice are widely used in biomedical research and due to its size the mouse brain is very suitable to study regional and cellular protein distributions in the mammalian nervous system. HPA antibodies against proteins expressed in the mouse nervous system are validated on mouse brain tissue using western blot and immunohistochemistry. 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.

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Bioinformatics

Group leader:

Kalle von Feilitzen

Personnel:

Dr Tove Alm (researcher), Dr Linn Fagerberg (researcher), Mattias Forsberg (research engineer), Fredric Johansson (research engineer), Dr Frida Henningson Johnson (researcher), Per Oksvold (research engineer), Lukas Persson (research engineer), Dr Åsa Sivertsson (researcher), Martin Zwahlen (research engineer)

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 Bioinformatics group performs computer-based analysis of protein sequences for selection of protein fragments (PrESTs) to be used as immunogens for generation of target-specific polyclonal antibodies. A custom made pipeline analyzes RNA-seq data from a large number of tissue, cancer and cell line samples. The group is also heavily involved in all research performed in the project, such as data collection, data interpretation and visualization as well as statistical analysis.

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Uppsala site

Site Director:Dr Cecilia Lindskog Bergström
Clinical Director:Prof Fredrik Ponten

ModuleGroup leader
Tissue Microarray Production, Immunohistochemistry, and ScanningIng-Marie Olsson
Antibody approval, Protein Profiling, and Antibody DestinyDr Cecilia Lindskog Bergström
Clinical PathologyProf Fredrik Ponten


Tissue Microarray Production, Immunohistochemistry, and Scanning

Group leader:

Ing-Marie Olsson (research engineer)

Personnel:

Maria Aronsson (research engineer), Dennis Kesti (biomedical analyst), Lillemor Källström (biomedical analyst)

Responsibility, TMA production:

(i) Production of tissue microarrays (TMAs) and cell microarrays (CMAs), (ii) handling of tissues (biobank material), cells and cell lines for TMA and CMA production and protein extraction, (iii) sectioning of tissue and TMA blocks, (iv) quality control of TMA-blocks, (v) maintenance and cultivation of cell lines and (vi) protein extraction for Western blot analysis.

Description, TMA production:

Tissue microarrays are used in the basic protein profiling for the Human Protein Atlas. Each protein expression profile is based on 8 TMAs including normal tissues from 144 different individuals (triplicate samples of 44 different tissue types) and cancer tissues from 216 different patients (duplicate samples of tumor tissues representing the 20 most common forms of human cancer).In total, 576 tissue cores are immunostained and analyzed for each antibody. The CMAs are used for antibody validation and include duplicate samples of 44 cell lines.

Formalin fixed, paraffin embedded tissue specimens are collected from the Department of Pathology, Uppsala University Hospital. Representative areas to include on the TMAs are defined for each tissue specimen by visual inspection of a corresponding hematoxylin-eosin stained section under a microscope. Tissue cores from these selected areas are then taken and used for TMA production. Cell lines are cultured in vitro, harvested, fixed in formalin and dispersed in agarose prior to embedding in paraffin blocks. These cell-blocks are used for the construction of CMAs.

Responsibility, Immunohistochemistry:

(i) Handling and storage of antisera, (ii) test and titration of antibodies for immunohistochemistry, and (iii) immunostaining of TMAs and CMAs.

Description, Immunohistochemistry:

Antibodies are titrated using a specially designed test-TMA, representing a limited selection of tissues and cells. The titration is performed based on stringent criteria, according to which different antibody dilutions are tested. The primary working dilution is based on the protein concentration for each antibody. When an optimal antibody dilution is found automated immunostaining is performed on sections from eight TMAs for basic protein profiling in the Human Protein Atlas project. Instruments and commercially available detection kits are used to ensure standardization and reproducibility of immunohistochemistry. In addition, protein extractions are prepared from fresh frozen tissues and cell lines for the purpose of running Western blot.

Responsibility, Scanning:

(i) Scanning of immunohistochemically stained TMA slides, (ii) export and processing of scanned digital images from TMAs, and (iii) administration of TMAx for automated image analysis.

Description, Scanning:

Immunostained TMA slides are scanned to generate high-resolution digital images, using 20x (tissue) or 40x (cells) magnification. Images from scanned TMAs are separated into spot images, representing immunostained tissue (1 mm diameter) or cell sample (0.6 mm diameter), and exported as individual TIFF files. Manual, pathology-based evaluation of images and annotation of protein expression in tissue is performed using an in-house built web based annotation software. Automated image-analysis algorithms using the TMAx system are used to generate protein expression data from images of immunostained CMAs.

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Antibody approval, Protein Profiling, and Antibody Destiny

Group leader:

Dr Cecilia Lindskog Bergström

Personnel:

, Groom Alemayehu (research engineer), Borbala Katona (research engineer), Feria Hikmet Noraddin (research engineer), Åsa Näsström (research engineer), Evelina Sjöstedt (research engineer), Jimmy Vuu (research engineer)

Responsibility:

(i) Validation of antibody target specificity (ii), maintenance of immunostaining reproducibility and quality, (iii) coordination and evaluation of antibodies submitted by commercial vendors and academic scientists, (iv) annotation and final approval of immunohistochemically stained normal and cancer tissues (v) generation of knowledge-based protein expression profiles, and (vi) continuous curation and quality assurance of protein profiles.

Description:

Optimal antibody dilution and target specificity is assessed by microscopical examination. Available information in public databases on gene, RNA and protein level, internal and external RNA-seq data, as well as internal technical validation, including independent antibodies, protein arrays and Western blots, are considered in the decision process. For each approved antibody, a final immunostaining protocol is defined and subsequently applied to the full-scale TMAs. The images are then scanned for generation of high-resolution digital images. Manual annotation of immunohistochemistry is performed by specially educated personnel using a web-based in-house developed software to record the intensity and fraction of immunoreactive cells for each given cell population, and to determine the subcellular localization of immunoreactivity. A text comment summarizing the characteristics for each antibody is added to the annotation. The results are visualized in a summary view as bars corresponding to the protein expression level in each given cell type. In total 76 normal cell types from 144 individuals and 20 different cancer cell types from 216 different tumors are annotated for each antibody. An independent second observer curates all finished annotations to ensure uniform annotations of high quality. After annotation and curation, protein profiles are evaluated and the generated data is compared with gene, RNA and protein characterization data. Antibodies that are approved at this stage are assigned a reliability score and a knowledge-based protein expression profile and released for publication in the next version of the Human Protein Atlas.

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Clinical Pathology

Group leader:

Prof Fredrik Ponten

Personnel:

Dr Julia Bergman (MD, PhD-student), Dijana Djureinovic (PhD-student)

Responsibility:

(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.

Description:

The HPA database is actively mined for potential biomarkers with the aim to identify protein expression patterns that could be of medical or biological significance. Projects include various forms of human disease with an emphasis on cancer. Most projects are focused on identification and validation of biomarker candidates that can fulfill currently unmet clinical needs related to diagnostics, prognostics and treatment prediction. To address such questions, patient cohorts representing different cancers are defined and tumor material as well as clinical data is collected. These specifically designed cancer TMAs are produced and used for extended analysis of protein expression patterns to test and validate candidate proteins as useful biomarkers. The biomarker discovery and validation efforts include both internal projects and external collaborative projects.

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