Protein quantification software
Determination of protein-protein interactions, proteome dynamics and differential expression are central issues of proteomics requiring reliable quantification. A number of different techniques, most of them based on metabolic or chemical labelling have been established recently. However, these are not readily applicable to native systems or large numbers of samples, and in addition suffer from a limited dynamic range. Thus, we have focused on improving sensitivity and linearity of label-free quantification using three complementary approaches: spectral counting, m/z peak volume determination and statistical evaluation. These methods are currently optimized and integrated into a novel quantification software for users of linear ion trap mass spectrometers.
We have specialized in proteomic analysis of small amounts of native tissue and continually seek to improve sensitivity and throughput. Our efforts focus on improving LC-MS/MS sensitivity, optimizing sample preparation and streamlining our PMAP technology. Current microproteomic benchmark projects comprise proteomic analysis of primary neuronal cells, identification of differential markers in solid tumor biopsies, and high-content profiling of antibodies.
The ability to efficiently purify native membrane target protein complexes opens new perspectives for drug development and screening: target-ligand interactions in a microdomain context, target selectivity profiles of small molecule ligands and screening of libraries for novel binders. We perform proof-of-principle studies for selected G-protein coupled receptors (including orphan receptors) and ion channels.
Protein interaction database
Up to now we have accumulated proteomic data from more than 1000 membrane protein target affinity purifications. These MS sequencing datasets form a valuable resource covering not only a significant portion of the brain proteome. They also contain detailed information about protein isoforms, modifications and association into complexes. We will develop bioinformatic tools to convert this information into a database that will fuel our internal development pipeline and may serve as a useful backup resource for future customers and partners.
Application projects with academic partners
Novel ion channel and receptor co-targets
We have used our proteomic technology to analyse ion channel and receptor complexes that play key roles in neurophysiology. This revealed a number of novel auxiliary subunits, for example of GABAB receptors, AMPA receptors, HCN channels, voltage-gated channels, that are good candidates for new drug targets. Together with academic collaboration partners, we will further study their physiological function and analyze their molecular structure.
Ca2+ is a key mediator of cellular signalling that controls synaptic transmission, plasticity and neuronal development in the central nervous system. Kinetics and topical/functional specificity are determined by intracellular Ca2+ nano- and microdomains that form around Ca2+ sources with co-localizing effectors and signalling modulators. To elucidate the molecular basis of neuronal calcium channel-associated microdomains, we have used a multi-epitope affinity purification strategy combined with knockout controls and quantitative mass spectrometry. Preliminary results indicate that Ca2+ channel-associated networks may comprise more than 100 proteins from different classes. We seek to further dissect these networks into sub-complexes and study their structure and function in collaboration with academic partners.
Pain pathway targets
The perception, transmission and processing of pain stimuli involves many ion channels and receptors that so far have not been characterized on a molecular level. Based on our microproteomics platform we have systematically analyzed targets in primary afferent neurons and spinal cord to elucidate their subunit composition and modulation pathways. We are further investigating candidates in heterologous systems with the goal to establish cell lines that reconstitute the physiological behaviour and provide a basis for meaningful screening of these challenging targets.
Analysis of mast cell signalling pathways
Mast cells are equipped with a number of membrane receptors that trigger and modulate immune responses and that have been implicated in asthma, inflammation and allergic reactions. We are studying the complex signalling pathways of immunoglobulin receptors, toll-like receptors, interleukin receptors and GPCRs to identify key mediators of specific immune signals.