Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

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, ϳ400,000 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.

Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci.

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor ␤ (PDGFR␤) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFR␤ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR␤ in porcine aortic endothelial cells transfected with the ␤-receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR␤. We furthermore visualized tyrosine phosphorylated PDGFR␤ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology. Molecular & Cellular Proteomics 6:1500 -1509, 2007.