Thermophoretic quantification of disease specific biomarkers in complex biological fluids
Final Report Abstract
Methods for determining personalized DNA markers and in particular, methods for personalized whole genome sequencing have advanced rapidly and the associated costs have declined at an unexpected pace. By contrast the methods for determining the levels of proteins in major human organ systems are much more expensive and challenging. Progress has recently been made with quantitative mass spectrometry, but the functions of each protein and their dependence on other variable proteins in the natural personal environment cannot be assessed by this technology. Clearly, mass spectrometry is a powerful, descriptive tool, but not suited to monitor the multitude of complex interactions on the cell surface, in the extracellular matrix and during cellular signaling and protease-mediated regulation. A limited number of functional assays are available for the most extensively studied protease cascade systems, the blood coagulation and complement systems. The proteases of these two systems, however, are only a small subset of more than 600 proteolytic enzymes in the human genome. Posttranslational modifications and biological regulation by proteases is based on finely tuned interactions between inhibitors, enzymes, and their respective substrates. The binding of an enzyme to a substrate is strongly influenced by the proteinaceous composition of the biological buffer system and by various as yet unknown plasma and matrix components. Moreover, interactions of the analyte with interfering factors from the personally unique, complex environment can modulate the binding to its functional targets, but are completely ignored by these methods. The funding allowed us to develop microscale thermophoresis significantly towards real world applications and high throughput screening. The partnership of this proposal allowed to develop microscale thermophoresis to a level where its use in blood plasma was demonstrated even under challenging biochemical conditions. We were able to shrink the volume usage from 0.5µl down to 10 nl in a water-in-oil immersion format in multi-well plates. In addition, we could demonstrate that even in complex and heterogeneous patient samples we can detect robustly a highly interesting, and statistically significant predictor for the strength of the disease. Taking a multidisciplinary approach we developed a quantitative thermophoresis method to analyze the initial rate-determining interaction of a decoy substrate, alpha-1- antitrypsin (AAT), with its target protease, neutrophil elastase, which subsequently traps the elastase by a covalent complex and removes it from the biological system. The clinically recognized manifestations of reduced AAT levels, lung emphysema, are caused by the relatively common Z-variant in 95% of all Caucasian cases, but susceptibility, onset and severity of lung disease cannot be predicted from AAT concentration measurements in homozygous carriers of the Z-mutation. The MST assay which we established, can detect both, changes of the concentration and the affinity of the analyte to its target in the person-specific environment and therefore characterizes the biological function of AAT in the person-specific proteome-wide context. The new parameter, the thermophoretic depletion amplitude, combines both parameters, affinity and concentration, in the natural plasma environment. Sample-dependent changes in the thermophoretic amplitude revealed unexpected interfering plasma factors never considered to date in studies on this interaction in standard buffers.
Publications
- Direct detection of antibody concentration and affinity in human serum using microscale thermophoresis. Analytical Chemistry 84, 3523–3530 (2012)
Svenja Lippok, Susanne A. I. Seidel, Stefan Duhr, Kerstin Uhland, Hans-Peter Holthoff, Dieter Jenne and Dieter Braun
(See online at https://doi.org/10.1021/ac202923j) - A monoclonal antibody (MCPR3-7) interfering with the activity of proteinase 3 by an allosteric mechanism. Journal of Biological Chemistry
Lisa C. Hinkofer, Susanne A. I. Seidel, Brice Korkmaz, Francisco Silva, Amber M. Hummel, Dieter Braun, Dieter E. Jenne, Ulrich Specks
(See online at https://doi.org/10.1074/jbc.M113.495770) - Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59:301–315 (2013)
Susanne A.I. Seidel, Patricia M. Dijkman, Wendy A. Lea, Geert van den Bogaart, Moran Jerabek-Willemsen, Ana Lazic, Jeremiah S. Joseph, Prakash Srinivasan, Philipp Baaske, Anton Simeonov, Ilia Katritch, Fernando A. Melo, John E. Ladbury, Gideon Schreiber, Anthony Watts, Dieter Braun, Stefan Duhr
(See online at https://doi.org/10.1016/j.ymeth.2012.12.005) - Thermophoresis in nanoliter-droplets quantifies aptamer binding. Angewandte Chemie 53, 7948–7951 (2014)
Susanne Seidel, Niklas Markwardt, Simon Lanzmich, Dieter Braun
(See online at https://doi.org/10.1002/anie.201402514) - Inhibitors and antibody fragments as potential anti-Inflammatory therapeutics targeting neutrophil proteinase 3 in human disease. Pharmacol Rev. 68:603-30 (2016)
Korkmaz B, Lesner A, Guarino C, Wysocka M, Kellenberger C, Watier H, Specks U, Gauthier F, Jenne DE
(See online at https://doi.org/10.1124/pr.115.012104)
