Tove Alm

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.

Engineering Bispecificity into a Single Albumin-Binding Domain

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.

A small bispecific protein selected for orthogonal affinity purification

A novel protein domain with dual affinity has been created by randomization and selection. The small alkali‐stabilized albumin‐binding domain (ABD*), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z2 domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A‐based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.

A Chromosome-Centric Analysis of Antibodies Directed toward the Human Proteome Using Antibodypedia

Antibodies are crucial for the study of human proteins and have been defined as one of the three pillars in the human chromosome-centric Human Proteome Project (C-HPP). In this article the chromosome-centric structure has been used to analyze the availability of antibodies as judged by the presence within the portal Antibodypedia, a database designed to allow comparisons and scoring of publicly available antibodies toward human protein targets. This public database displays antibody data from more than one million antibodies toward human protein targets. A summary of the content in this knowledge resource reveals that there exist more than 10 antibodies to over 70% of all the putative human genes, evenly distributed over the 24 human chromosomes. The analysis also shows that at present, less than 10% of the putative human protein-coding genes (n = 1882) predicted from the genome sequence lack antibodies, suggesting that focused efforts from the antibody-based and mass spectrometry-based proteomic communities should be encouraged to pursue the analysis of these missing proteins. We show that Antibodypedia may be used to track the development of available and validated antibodies to the individual chromosomes, and thus the database is an attractive tool to identify proteins with no or few antibodies yet generated.

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 . The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

Introducing the Affinity Binder Knockdown InitiativeA publicprivate partnership for validation of affinity reagents

Graphical abstract