The Executive Management is responsible for the overall strategies of the consortium and for managing the overall program:
Prof Mathias Uhlen, Program Director
Prof Fredrik Ponten, Vice Program Director
Prof Sophia Hober, Director of Protein Science
Assoc prof Caroline Kampf, Site Director Uppsala
Dr Jenny Ottosson Takanen, Site Director AlbaNova, Stockholm
Prof Peter Nilsson, Site Director SciLifeLab, Stockholm
Dr Lisa Björling, Director of Bioinformatics
Dr Emma Lundberg, Director for Subcellular Atlas
Dr Sanjay Navani, Site Director Mumbai, India
The Scientific Advisory Board (SAB) consists of representatives mainly from the academic field in areas
such as bioinformatics, protein science and cell biology. The role of the SAB is to evaluate the scientific
out-put and to advise in new developments and external collaborations.
Dr Cristina Al-Khalili Szigyarto is responsible for external relations.
AlbaNova site, Stockholm (site director: Jenny Ottosson Takanen)
SciLifeLab site, Stockholm (site director: Peter Nilsson)
Uppsala site (site director: Caroline Kampf)
Lab SurgPath, Mumbai site (site director: Sanjay Navani)
AlbaNova site, Stockholm
| Site Director: |
Dr Jenny Ottosson Takanen |
Bioinformatics
Group leader: Dr Lisa Björling
Personnel: Dr Linn Fagerberg (researcher), Mattias Forsberg (research engineer), Kalle Jonasson (research engineer),
Per Oksvold (research engineer), Dr Åsa Sivertsson (researcher) and 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, and (iii) to initiate the analysis of human
proteins by in silico selection of Protein Epitope Signature Tags (PrESTs) with subsequent design of oligonucleotide primer
pairs for the cloning procedures in the Molecular Biology module.
Description: With the LIMS (Laboratory Information Management System) as the backbone, data is collected from each
module in the pipeline. The protein expression profiles 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. The group is also heavily involved in all
research performed in the project, such as data collection, data interpretation and statistical analysis.
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Molecular Biology
Group leader: Holger Eklund
Personnel: Malin Cammenberg (research engineer), Jenny Fall (technician),
Lili Gong (technician), Marica Hamsten (research engineer) and Mehri Salahi (technician).
Responsibility: Production of recombinant PrEST expression clones including cDNA synthesis, cloning, and
plasmid purification. All clones are quality controlled with DNA sequencing.
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. 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 an albumin binding domain. After transformation into E. coli
Rosetta, 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 produced for
delivery to the Protein Factory module.
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Protein Factory
Group leader: Hanna Tegel
Personnel: Anna Bäckström (research engineer), Adrian Gronowski (technician), Anneli Halldin (technician), Sebastian Johansson (technician), Katarina Malm (research engineer), Axel Riese (technician) and Lan Lan Xu (laboratory engineer).
Responsibility: To express and purify PrEST-proteins to use for the preparation of antigens and affinity columns.
Description: The Protein Factory group is responsible for protein expression and purification in the Human Protein
Atlas program. 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, the
Protein Factory produces affinity resin with His6ABP-ligand.
Abbreviations:
ABP - Albumin Binding Protein
IPTG - Isopropyl-B-D-Thiogalactopyranoside
IMAC - Immobilized Metal Affinity Chromatography
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Immunotechnology
Group leader: Cajsa Älgenäs
Personnel: Annelie Cajander (research engineer), Andreas Hober (technician), Karin Jakobsson (laboratory engineer), Anna Johansson (technician) and Cecilia Mikaelsson (laboratory engineer).
Responsibility: (i) Management of immunization outsourcing, (ii) Generation of monospecific antibodies through affinity purification of polyclonal antisera, (iii) Western blot (WB) analysis of antibodies approved in protein array analysis, (iv) WB antibody validation using over-expression lysates, (v) storage and distribution of antibodies within the program and to collaborative partners.
Description: The Immunotechnology group is 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% glycerol and 0,02% 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(TM), OriGene Technologies, Rockville, MD) as a positive control.
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Epitope Mapping and Therapeutic Antibodies
Group leader: Dr Johan Rockberg
Personnel: Björn Forsström (PhD-student), Barbara Hjelm (PhD-student), Dr Paul Hudson (post-doc), Francis Jingxin Hu (PhD-student), Dr Andrej Nikosjkov (Senior scientist), Dr Helena Persson (senior scientist) and Anna-Luisa Volk (Master student).
Responsibility: (i) To determine antibody epitopes for HPA antibodies (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 (iii) Large scale synthetic peptide arrays.
Monoclonal antibodies to clinically relevant target proteins are generated as hybridomas, screened for desired effect in cell-based assays and
cloned for humanization and affinity maturation.
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Protein Science
Group leader: Prof Sophia Hober
Personnel: Dr Anja Persson (project leader), Anna Konrad (PhD-student), Johan Nilvebrant (PhD-student), Mikael Åstrand (PhD-student), Tove Boström (PhD-student) and Dr Carl Hamsten (post-doc).
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, localisation and interactions of human proteins.
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SciLifeLab site, Stockholm
| Site Director: |
Prof Peter Nilsson |
Protein Array Technologies
Group leader: Prof Peter Nilsson
Personnel: Burcu Ayoglu (PhD-student), Julie Bachmann (postdoc), Sanna Byström (PhD-student), Spyros Darmanis (postdoc), Mun-Gwan Hong (postdoc), Ida Hossar (research engineer), Anna Häggmark (PhD-student), Ulrika I Qundos (PhD-student), Maja Neiman (PhD-student), Ronald Sjöberg (research engineer) and Assoc prof Jochen M. Schwenk (researcher)
Responsibility: To analyse the specificity and quality of all purified HPA antibodies, develop and utilize antigen
and antibody based microarray methodologies for large scale analysis of bodyfluids.
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: Dr Emma Lundberg
Personnel: Hammou Ait Blal (research engineer), Frida Danielsson (PhD-student), Martin Hjelmare (research engineer), Dr Elton Rexhepaj (post-doc), Marie Skogs (PhD-student), Charlotte Stadler (PhD-student), Diana Telessemian (research engineer), Mikaela Wiking (research engineer), Ulla Wrethagen (technician) and Annika Åbergh (technician).
Responsibility: To analyze the subcellular distribution of proteins, including; (i) immunofluorescent staining of cell lines, (ii) confocal imaging of immunofluorescently stained samples, (iii) annotation of subcellular staining patterns, (iv) knowledge-based curation of the subcellular distribution in a gene-centric manner.
Description: The cell profiling module is responsible for the subcellular localizations of proteins using antibodies generated in the Human Protein Atlas (HPA) program.
Three cell lines are cultured in vitro, fixated/permeabilized using formaldehyde/detergent treatment and immunofluorescently stained according to standardized protocols. Besides the HPA antibodies, the cells are stained with two organelle markers specific for the endoplasmic reticulum and microtubules, as well as counterstained with the nuclear probe DAPI.
Two high-resolution, four-color images, are acquired per antibody and cell line. The images are acquired manually using Leica SP5 confocal laser scanning microscopes equipped with a 63x magnification, oil immersion objective. The microscope operator ensures that the acquired images are representative for the entire sample and positions the optical section at an appropriate level.
All images are manually annotated and for each image pair the subcellular localization, characteristics and intensity of the staining is described. Further, a validation score is set for each antibody indicating if the results are supported by other experimental data. 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. Here, the subcellular distribution is classified as main and additional locations.
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Plasma Profiling
Group leader: Assoc prof Jochen M. Schwenk
Personnel: Kimi Drobin (research engineer), Ulrika I Qundos (PhD student), Maja Neiman (PhD student), Burcu Ayoglu (PhD student), Anna Häggmark (PhD student), Sanna Byström (PhD student), Spyros Darmanis (Postdoc), Mun-Gwan Hong (Postdoc), Julie Bachmann (Postdoc) and Prof Peter Nilsson (researcher)
Responsibility: Utilizing HPA antibodies in multiplexed affinity arrays for protein profiling of human serum/plasma for the discovery, identification and verification potential biomarker candidates.
Description: Antibody suspension bead arrays are employed to protein profile biotinylated serum/plasma samples across diseases, age and gender. Each month, purified and validated antibodies are immobilized on fluorescently color-coded beads to create new series of bead arrays, which are composed of 384 antibodies each. During a discovery phase, this microtiter plate based procedure is used to explore the protein profiles within a multi-disease cohort, which comprises 24 different diseases such as cancer, cardiovascular and neurodegenerative diseases. Significant differences are identified using statistical models and disease/protein associations are verified regarding technical and biological aspects. Subsequently, a focus is set to screen dedicated single-disease cohorts with larger numbers of specimen from independent collections to ultimately suggest target proteins for the development of sandwich immunoassays. Besides this, a study is ongoing to profile proteins in serum/plasma in a cohort composed of males and females between 5-85 years of age that were without a diagnosed disease at the time point of sample collection. Currently, we are also developing protocols for the analysis of additional body fluids such as cerebrospinal fluid or urine.
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Brain Profiling
Group leader: Dr Jan Mulder
Personnel: Tony Jimenez-Beristain (technician) and Dr Nicholas Mitsios (researcher)
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 30-35 coronal sections of the mouse brain with a 400 µm section interval covering all major brain nuclei. Whole slide immunofluorescence captured at 20x 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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Uppsala site
| Site Director: | Assoc prof Caroline Kampf |
Tissue Microarray Production, Sectioning and Scanning
Group leader: Ing-Marie Olsson (technician)
Personnel: Eléne Carlberg (technician), Sofie Gustafsson (biomedical analyst), Frank Hammar (biomedical analyst) and Ann-Sofi Strand (biomedical analyst).
Responsibility: (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) scanning of immunohistochemically stained TMA slides, (vi) export and processing of scanned digital images from TMAs, and (vii)
administration of TMAx for automated image analysis.
Description:Formalin fixed and paraffin embedded tissue specimens are collected from the Department of Pathology, Uppsala University
Hospital. Areas of representative tissue are defined from a corresponding hematoxylin-eosin stained tissue section through visual inspection under
the microscope. 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.
Basic protein profiling to determine protein expression patterns for the Human Protein Atlas is based on 8 TMAs and 1 CMA. In total 708 spots of
tissues and cells are immunostained and analyzed for each antibody. The TMAs include normal tissues from 144 different individuals (triplicate
samples of 48 different tissue types) and cancer tissues from 216 different patients (duplicate samples of tumor tissues representing the 20 most
common forms of human cancer). The CMA includes duplicate samples of 47 cell lines and 12 clinical cell samples.
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 a web based
annotation software. Automated image-analysis algorithms using the TMAx system is used to generate protein expression data from images of immunostained
CMAs.
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Immunohistochemistry
Group leader: Urban Ryberg (technician)
Personnel: Dijana Cerjan (biomedical analyst), Åsa Edvinsson (technician), Veronica Lundström (biomedical analyst) and Eva Wahlund (biomedical analyst).
Responsibility: (i) Handling and storage of antisera, (ii) test and titration of antibodies for immunohistochemistry, (iii) immunostaining of
TMAs, (iv) protein extraction for Western blot analysis and (v) Western blot analysis.
Description: Antibodies are titrated using a specially designed test-TMA, representing a limited selection of tissues and cells. The titration
is done according to a defined schedule, according to which different antibody dilutions and antigen retrieval methods are tested. The primary working
dilution is based on the protein concentration for each antibody. Automated immunostaining is performed on sections from 8 TMAs and one CMA 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 Western blotting. Western
blots are performed on selected antibodies.
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Antibody Approval and Annotated Protein Expression
Group leader: Evelina Sjöstedt (technician)
Personnel: Dijana Djurenovic (technician), Hanna Emanuelsson (technician), Jerker Linné (technician), John Juter (technician), Linda Oskarsson (technician), Jennie Pettersson (technician), Marcus Runeson (technician) and Stina Sundström (technician).
Responsibility: (i) Validation of antibody target specificity (ii), maintenance of immunostaining reproducibility and quality, (iii) coordination of
evaluation of antibodies submitted by commercial vendors and academic scientists, (iv) evaluation of protein profiles, (v) generation of annotated protein expression
profiles.
Description: Optimal antibody dilution and target specificity is assessed by microscopical examination. Available information both from gene and protein
public databases, as well as in-house technical validation, such as 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.
Protein profiles from TMAs are evaluated and approved antibodies are assigned a reliability score and released for publication in the next version of the Human
Protein Atlas. An annotated protein expression is performed for all proteins that have two or more corresponding antibodies. The antibody-based data is merged taking
into account the performance of respective antibodies and a review of available protein/gene characterization data to obtain a best estimate of expression pattern.
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Biomarker Discovery
Group leader: Dr Per-Henrik Edqvist
Personnel: Jerker Linné (technician) and Marcus Runeson (technician).
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, particularly cancer, (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 indicate whether a
particular protein could be used as a biomarker for diagnostics, prognostics or treatment prediction for a given type of cancer. The focus is to identify and
validate biomarker candidates that can fulfill currently unmet clinical needs in cancer diagnostics and oncology. Examples of unmet clinical needs we work to meet
are biomarkers that stratify patient groups into high and low risk for recurrence, responders and non-responders to a given therapeutic regime, benefits and side effects
from surgery versus watchful waiting. 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 prognostic and/or predictive cancer biomarkers. The biomarker discovery effort includes both internal projects and external collaborative projects.
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Technical Development
Group leader: Dr Anna Asplund
Personnel: Sandra Andersson (PhD-student), Dr Angelika Danielsson (post-doc), Gabriela Gremel (post-doc), Cecilia Lindskog (PhD-student), Kristina Magnusson (PhD-student), Elton Rexhepaj (post-doc) and Dr Kenneth Wester (researcher).
Responsibility: (i) To develop and set up of methods for validation and characterization of antibodies and their performance, and (ii) to constitute a technical
resource for external and internal research projects.
Description: The HPA continuously uses antibodies as protein probes for both large scale protein expression mapping in human normal tissues and cancer, and for a
number of external and internal research projects. Methods generating a greater understanding of antibody performance and characteristics constitute a valuable resource in
both pursuits. Technical development aims to expand the panel of methods at hand at the Uppsala site, to include for example siRNA knock-down of expression, PLA for
brightfield microscopy, LigandTracer analysis of binding kinetics, functional analyses in cell lines and image analysis of protein expression in both tissues and cells.
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Lab SurgPath, Mumbai site
| Site Director: | Dr Sanjay Navani |
Pathology-based Annotation
Group leader: Dr Sanjay Navani
Personnel: Dr Naila Khan (research associate), Dr Tushar Patil (research associate), Nitin Pardule (technician) and Avadhesh Vishwakarma
(IT administrator)
Responsibility: (i) Organization and administration of pathology-based annotation and curation of immunohistochemistry-based protein profiling in normal
and cancer tissues, (ii) recruitment and training of personnel for the HPA-Mumbai site, (iii) initiation, supervision and collaboration for India-based research
projects.
Description: Manual annotation of immunohistochemistry is performed on high-resolution images generated in the tissue microarray module. A web-based
in-house developed software is used 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 color codes corresponding to the protein expression level in each given cell type. In total 67 normal cell types from 144 individuals and 20
different cancer cell types from 216 different tumors are annotated for each antibody. An independent pathologist or specially trained personnel curates all
finished annotations to ensure uniform annotations of high quality.
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