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Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p

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.

Advances in antibody production render a growing supply of affinity reagents for immunohistochemistry (IHC), and tissue microarray (TMA) technologies facilitate simultaneous analysis of protein expression in a multitude of tissues. However, collecting validated IHC data remains a bottleneck problem, as the standard method is manual microscopical analysis. Here we present a high-throughput strategy combining IHC on a recently developed cell microarray with a novel, automated image-analysis application (TMAx). The software was evaluated on 200 digital images of IHC-stained cell spots, by comparing TMAx annotation with manual annotation performed by seven human experts. A high concordance between automated and manual annotation of staining intensity and fraction of IHC-positive cells was found. In a limited study, we also investigated the possibility to assess the correlation between mRNA and protein levels, by using TMAx output results for relative protein quantification and quantitative real-time PCR for the quantification of corresponding transcript levels. In conclusion, automated analysis of immunohistochemically stained in vitro-cultured cells in a microarray format can be used for high-throughput protein profiling, and extraction of RNA from the same cell lines provides a basis for comparing transcription and protein expression on a global scale.