Mass Photometer-Mass Fluidics High Concentration System

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 550111075

The aim of this application is to enable the routine, fast, and precise investigation of unlabeled biopolymers - proteins and DNA - in solution using mass photometry (MP). MP measures the mass of particles (biopolymers) of a molecular size between 30 kDa and 5 MDa freely in solution in vitro. This mass range is suitable for many proteins, DNAs, and their complexes. MP detects light that gets scattered by particles landing onto a glass surface from solution. Similar to the physiological situation, biopolymers do not need to carry chemical modifications or be immobilized on a surface. MP has a resolution of approximately 2% of the mass to be determined. In the proposed form (including the mass photometer-mass fluidics high concentration system), MP operates in the nM-µM concentration range, where many binding reactions of biopolymers occur. MP is therefore suitable for complex formation studies. This proposal involves chemical and molecular biology research groups. The molecular biologists aim to use MP to understand the formation and regulation of protein complexes that play central roles in the inheritance of the genetic information. This includes studying protein complexes that mediate and regulate mitotic chromosome distribution and DNA replication. Another goal is to characterize protein complexes that help maintain the physiological balance of cellular proteins (proteostasis). The chemists aim to produce DNA nanostructures using DNA origami with support of MP and to understand the effects of biologically active small molecules. Additionally, they are developing small molecules for targeted protein degradation in cells (PROTACs). For these objectives, MP is used by the biologists for the - partially quantitative - characterization of protein and DNA binding reactions. The regulation of these reactions by protein kinases is also investigated. The chemists will use MP to produce and test DNA-DNA and DNA-protein complexes as building blocks for larger nanostructures (DNA origami). Furthermore, they will use MP to understand the effects of bioactive small molecules on protein complex formation. In these studies, MP will partly replace and partly complement techniques already in use. The advantages of MP lie in its resource-efficiency compared to previously used size exclusion chromatography and isothermal calorimetry. Compared to classical affinity pulldown experiments and surface plasmon resonance measurement, MP does not require modifications such as protein tags and immobilization of biopolymers. The rapid, resource-efficient MP also allows routine applications for optimizing buffer conditions and quality testing of protein preparations.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Massenphotometer-Massendurchfluss-Hochkonzentration System
• Instrumentation Group: 5360 Meßgeräte für gestreutes und reflektiertes Licht, optische Oberflächen-Prüfgeräte
• Applicant Institution: Universität Duisburg-Essen
• Leader: Professor Dr. Dominik Boos