DigiWest: a high throughput Western-Blot and its application for comprehensive signaling analysis of microdissected liver tissue

Proteins play a pivotal role in cellular processes. As proteins represent the major ‘workforce’ of biomolecules, changes in protein abundances and in posttranslational modifications indicate changes in the behavior of cells on the molecular level. Analysis of proteins improves our understanding of physiological and pathophysiological mechanisms. Today, there are numerous technologies and methods available for protein analysis. For semi-quantitative detection, Western-Blotting still represents the gold standard and is probably the most widespread method for protein analysis. The DigiWest approach which is described in this thesis is based on the Western-Blot. Transfer onto a bead-based microarray platform creates hundreds of replicas of an initial blot. Proteins are separated by SDS-PAGE and blotted onto a membrane as performed for a classical Western-Blot. The membrane containing the size-separated proteins is then cut into molecular weight fractions. Proteins are eluted from the molecular weight fractions and each fraction is loaded onto a distinct color-coded Luminex bead set. The color code of the bead sets retains the molecular weight information and allows pooling of all bead sets loaded with proteins into a bead-mix which represents the initial Western-Blot with a resolution of 0.5 mm. As only a small aliquot of a bead-mix is required for an antibody-incubation, a bead-mix loaded with protein from a single Western-Blot is sufficient for hundreds of antibody incubations which are performed in microtiter plates. After fluorescence-based readout on the Luminex instrument, the color code of the beads allows digital reconstruction of the initial Western-Blot lanes using the antibody specific signal obtained on the different bead-sets representing molecular weight fractions of the initial blot. Therefore, the method was named ‘DigiWest’. The digital data allow fast quantification of the antibody specific signal without image processing and the data can also be used to create gray-scale images mimicking classical Western-Blot images. Although hundreds of replicas are generated, allowing hundreds of antibody incubations in fast and automatable assays, DigiWest uses the same sample amount that is usually used for a single Western-Blot while it keeps sensitivity and signal linearity comparable to high-end Western-Blot readout platforms. The results are robust and highly comparable to classical Western-Blots. Application of the DigiWest method allowed a comprehensive analysis of laser capture microdissected samples which are barely sufficient for a single Western-Blot. Mouse models play an important role in toxicology and the liver, the most important organ for drug metabolism, gains special attention. Besides its homogenous appearance the liver is built from lobules as repeated units and zonation results in different metabolic competences for the hepatocytes dependent on their position within a lobule. Proximal periportal and pericentral zones from liver lobules were isolated by LCM from formalin fixed mouse liver sections derived from a time-course treatment study with the non-genotoxic carcinogen TCPOBOP. About 200 Western-Blot equivalents were performed with the resulting samples. The analysis of the isolated zones provided new insights into cellular signaling in liver zonation and changes occurring during the treatment time course. An important role for serine and threonine phosphorylations and phosphatases was found for regulation of zone specific metabolism. Inhibition of the phosphatase PP2A in the periportal zone was found to direct insulin dependent signaling. The TCPOBOP treatment time-course analysis showed that mainly the pericentral zone is exposed to oxidative stress resulting in activation of a stress-survival response while other TCPOBOP induced effects were found to affect both zones. TCPOBOP was found to induce a pronounced disturbance in cellular signaling within the first 24 hours after treatment

Investigating Complex Samples with Molograms of Low-Affinity Binders

In vitro diagnostics relies on the quantification of minute amounts of a specific biomolecule, called biomarker, from a biological sample. The majority of clinically relevant biomarkers for conditions beyond infectious diseases are detected by means of binding assays, where target biomarkers bind to a solid phase and are detected by biochemical or physical means. Nonspecifically bound biomolecules, the main source of variation in such assays, need to be washed away in a laborious process, restricting the development of widespread point-of-care diagnostics. Here, we show that a diffractometric assay provides a new, label-free possibility to investigate complex samples, such as blood plasma. A coherently arranged sub-micron pattern, that is, a peptide mologram, is created to demonstrate the insensitivity of this diffractometric assay to the unwanted masking effect of nonspecific interactions. In addition, using an array of low-affinity binders, we also demonstrate the feasibility of molecular profiling of blood plasma in real time and show that individual patients can be differentiated based on the binding kinetics of circulating proteins.ISSN:2379-369

An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced "cell-based molography" as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells