Johan Rockberg

Tissue-based map of the human proteome

Protein expression across human tissues Sequencing the human genome gave new insights into human biology and disease. However, the ultimate goal is to understand the dynamic expression of each of the approximately 20,000 protein-coding genes and the function of each protein. Uhlén et al. now present a map of protein expression across 32 human tissues. They not only measured expression at an RNA level, but also used antibody profiling to precisely localize the corresponding proteins. An interactive website allows exploration of expression patterns across the human body. Science, this issue 10.1126/science.1260419 Transcriptomics and immunohistochemistry map protein expression across 32 human tissues. INTRODUCTION Resolving the molecular details of proteome variation in the different tissues and organs of the human body would greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on quantitative transcriptomics on a tissue and organ level combined with protein profiling using microarray-based immunohistochemistry to achieve spatial localization of proteins down to the single-cell level. We provide a global analysis of the secreted and membrane proteins, as well as an analysis of the expression profiles for all proteins targeted by pharmaceutical drugs and proteins implicated in cancer. RATIONALE We have used an integrative omics approach to study the spatial human proteome. Samples representing all major tissues and organs (n = 44) in the human body have been analyzed based on 24,028 antibodies corresponding to 16,975 protein-encoding genes, complemented with RNA-sequencing data for 32 of the tissues. The antibodies have been used to produce more than 13 million tissue-based immunohistochemistry images, each annotated by pathologists for all sampled tissues. To facilitate integration with other biological resources, all data are available for download and cross-referencing. RESULTS We report a genome-wide analysis of the tissue specificity of RNA and protein expression covering more than 90% of the putative protein-coding genes, complemented with analyses of various subproteomes, such as predicted secreted proteins (n = 3171) and membrane-bound proteins (n = 5570). The analysis shows that almost half of the genes are expressed in all analyzed tissues, which suggests that the gene products are needed in all cells to maintain “housekeeping” functions such as cell growth, energy generation, and basic metabolism. Furthermore, there is enrichment in metabolism among these genes, as 60% of all metabolic enzymes are expressed in all analyzed tissues. The largest number of tissue-enriched genes is found in the testis, followed by the brain and the liver. Analysis of the 618 proteins targeted by clinically approved drugs unexpectedly showed that 30% are expressed in all analyzed tissues. An analysis of metabolic activity based on genome-scale metabolic models (GEMS) revealed liver as the most metabolically active tissue, followed by adipose tissue and skeletal muscle. CONCLUSIONS A freely available interactive resource is presented as part of the Human Protein Atlas portal (www.proteinatlas.org), offering the possibility to explore the tissue-elevated proteomes in tissues and organs and to analyze tissue profiles for specific protein classes. Comprehensive lists of proteins expressed at elevated levels in the different tissues have been compiled to provide a spatial context with localization of the proteins in the subcompartments of each tissue and organ down to the single-cell level. The human tissue–enriched proteins. All tissue-enriched proteins are shown for 13 representative tissues or groups of tissues, stratified according to their predicted subcellular localization. Enriched proteins are mainly intracellular in testis, mainly membrane bound in brain and kidney, and mainly secreted in pancreas and liver. Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray–based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.

A Human Protein Atlas for Normal and Cancer Tissues Based on Antibody Proteomics

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ∼400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Epitope mapping of antibodies using bacterial surface display

We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

High-resolution Mapping of Linear Antibody Epitopes Using Ultrahigh-density Peptide Microarrays

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.

Selection of protein epitopes for antibody production

Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.

Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibodys linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.

Contribution of Antibody-based Protein Profiling to the Human Chromosome-centric Proteome Project (C-HPP)

A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas ( www.proteinatlas.org ).

A whole‐genome bioinformatics approach to selection of antigens for systematic antibody generation

Here, we present an antigen selection strategy based on a whole‐genome bioinformatics approach, which is facilitated by an interactive visualization tool displaying protein features from both public resources and in‐house generated data. The web‐based bioinformatics platform has been designed for selection of multiple, non‐overlapping recombinant protein epitope signature tags by display of predicted information relevant for antigens, including domain‐ and epitope sized sequence similarities to other proteins, transmembrane regions and signal peptides. The visualization tool also displays shared and exclusive protein regions for genes with multiple splice variants. A genome‐wide analysis demonstrates that antigens for approximately 80% of the human protein‐coding genes can be selected with this strategy.

Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes

As antibody-based diagnosis and therapy grow at an increased pace, there is a need for methods which rapidly and accurately determine antibody-antigen interactions. Here, we report a method for the multiplex determination of antibody epitopes using bacterial cell-surface display. A protein-fragment library with 107 cell clones, covering 60 clinically-relevant protein targets, was created and characterized with massively parallel sequencing. Using this multi-target fragment library we determined simultaneously epitopes of commercial monoclonal and polyclonal antibodies targeting PSMA, EGFR, and VEGF. Off-target binding was observed for one of the antibodies, which demonstrates the method´s ability to reveal cross-reactivity. We exemplify the detection of structural epitopes by mapping the therapeutic antibody Avastin. Based on our findings we suggest this method to be suitable for mapping linear and structural epitopes of monoclonal and polyclonal antibodies in a multiplex fashion and could find applicability in serum profiling as well as other protein-protein interaction studies.

Staphylococcal Surface Display in Combinatorial Protein Engineering and Epitope Mapping of Antibodies

The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.