Noncovalent Interactions

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Since the pioneering work of J.J. Thomson, MS has become a centerpiece technology for characterization of molecules and detailed structure elucidation. In our opinion, no other analytical tool has experienced such a tremendous growth in its uses. This has largely been catalyzed by the advent of soft ionization methods in MS, namely matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) which enabled the ionization of large (>10 kDa) biomolecules without fragmentation. In recent years these techniques have become increasingly more useful to study biomolecules in their so-called,,native like“ states. This has been realized by either “native electrospray ionization” or chemical crosslinking followed by high mass MALDI-MS analysis.

In the electrospray ionization side, we are working on a better understanding of the fundamentals for the detection of non-covalent complexes with ESI-MS. Because, it is still under debate if and for which conditions ESI-MS gives accurate and reliable snapshot of solution phase chemistry of biomolecules.

The detection of large non-covalent protein complexes has also been successfully realized in our group by combining chemical cross-linking with a high mass MALDI-ToF MS. Non-covalent interactions of complexes are first stabilized with “chemical glues” (cross-linkers) and detected much more efficiently by the high mass sensitive ion conversion dynode (ICD) technology.

We apply both native ESI-MS and chemical crosslinking combined with high mass MALDI to approach various biochemical problems to get more analytical, biophysical and biological insights.

A high mass ion conversion dynode for MALDI experimetns and a nano electrospray ionization are shown as distingued workflows for the analysis of noncovalent interactions.   
Figure 1: Schematical representation of MS approaches to study non-covalent interactions

Current Scientific Projects

Fragment-Based Drug Discovery using nanoESI-MS. An emerging approach for generating hit compounds is fragment-based drug discovery (FBDD). This method involves the examination of small fragments that are synthetically modified into lead compounds by iterative cycles of synthesis. The noncovalent interactions of the fragments are weak and they become more potent in the fragment elaboration stage. Despite the advantages of ESI-MS over the existing biophysical methods for measuring binding affinities, it has not been used in FBDD. NanoESI-MS is used for determining binding affinities of a series of inhibitors for investigating protein-protein interactions and tumor growth proteins.

Investigation of Membrane Protein Interactions via High Mass MALDI-ToF XL-MS. In previous studies, chemical cross-linking combined with high mass MALDI-MS has been successfully applied in numerous systems, as antigen-antibody interactions, hemoglobin complexes, and hormone receptor complexes. Applying this method to intact membrane complexes is still challenging due to the inherent solubility and dynamic properties of membrane proteins as well as the low abundance and the absence of polar side chains in amino acid residues."

Surface plasmon resonance imaging (SPRi) coupled with MALDI MS (SPRi-MALDI). In clinical research and diagnostics exist high interest for more accurate and targeted determination of biomarkers. Surface plasmon resonance (SPR) is suitable to analyze biomolecules in a label-free fashion, and provides information on binding kinetics (kon/koff) and binding affinity in real time. Rapid and high-throughput analysis of different interactions in parallel can be achieved by working in an array format (SPR imaging). Coupling SPRi with MALDI MS enables a multiplexed detection and quantification of interactions by SPRi on one hand, and on the other hand, identification of interacting ligands by MALDI MS direct on chip.

Gap Sampler. Among analytical tools for the early drug discovery process, direct hyphenation of miniaturized sampling devices to electrospray ionization mass spectrometry (ESI-MS) is attractive. Reasons are that ESI-MS is compatible with microfluidics, and allows comprehensive, label-free sample analysis yielding information that is orthogonal to that available from optical methods.We present a “capillary gap sampler” as a platform for directly connecting microfluidics to ESI-MS. As shown in the figure the core of the sampler consists of a liquid bridge of several nanoliters formed within a µm-sized gap of two capillaries, whereas one of the capillaries acts directly as the ESI-MS interface. A solid pin is used for site-specific sample transfer with micrometer resolution. The sampler allows fast sample introduction of a few nanoliters into a controlled liquid bridge as a new microfluidic element.

Staff: Agni Gavriilidou, Katharina Root, Martin Köhler, Prince TiwariSahar Ghiasikhou, Ulrike Anders, Dr. Martin Czár

 
Page URL: http://www.zenobi.ethz.ch/research/noncovalent-interactions.html
29.05.2017
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