Periodic Reporting for period 2 - Top-AMPK (Putting the top down on AMPK protein complex)
Reporting period: 2024-08-01 to 2026-01-31
Proteins rarely act alone. They form complexes, carry chemical modifications, and exist as multiple distinct proteoforms that together determine cellular function. Understanding this molecular complexity is a prerequisite for advancing personalized medicine, yet the tools to characterize it at sufficient resolution have only recently become available.
Modern advances enable treatments increasingly tailored to an individual's molecular profile through greater precision in diagnosis, therapy, and prognosis. This approach aims to address disease causes rather than symptoms, with the potential for more targeted treatments with fewer side effects compared to conventional approaches. Achieving this requires analytical methods capable of resolving the full chemical heterogeneity of disease-relevant proteins, including modification states and proteoform distributions that previously remained inaccessible to conventional approaches.
This project focused on the 5'-adenosine monophosphate-activated protein kinase (AMPK), known as the guardian of cellular energy, a central regulator of cellular energy metabolism operating through complex phosphorylation networks, and associated with metabolic disorders, cardiovascular diseases, and viral infections. Native top-down mass spectrometry was applied as the core methodology, allowing intact protein complexes to be analyzed while preserving non-covalent interactions and capturing the full proteoform landscape.
The project brought together three complementary partners. The outgoing phase was carried out at the University of Wisconsin-Madison, chosen for its strong expertise in top-down proteomics, cardiac research, and kinase biology. The return phase was hosted at the University of Lübeck and CSSB Hamburg, providing a research environment centered on structural biology and native mass spectrometry. The non-academic placement at Bruker Daltonics connected the academic research directly to mass spectrometry instrument development, enabling knowledge transfer in both directions between fundamental research and industrial application.
The broader objective was to establish a methodology applicable beyond AMPK, contributing to the molecular characterization of disease-relevant kinase complexes and illustrating how fundamental research in structural proteomics can inform future clinical development.
Modern advances enable treatments increasingly tailored to an individual's molecular profile through greater precision in diagnosis, therapy, and prognosis. This approach aims to address disease causes rather than symptoms, with the potential for more targeted treatments with fewer side effects compared to conventional approaches. Achieving this requires analytical methods capable of resolving the full chemical heterogeneity of disease-relevant proteins, including modification states and proteoform distributions that previously remained inaccessible to conventional approaches.
This project focused on the 5'-adenosine monophosphate-activated protein kinase (AMPK), known as the guardian of cellular energy, a central regulator of cellular energy metabolism operating through complex phosphorylation networks, and associated with metabolic disorders, cardiovascular diseases, and viral infections. Native top-down mass spectrometry was applied as the core methodology, allowing intact protein complexes to be analyzed while preserving non-covalent interactions and capturing the full proteoform landscape.
The project brought together three complementary partners. The outgoing phase was carried out at the University of Wisconsin-Madison, chosen for its strong expertise in top-down proteomics, cardiac research, and kinase biology. The return phase was hosted at the University of Lübeck and CSSB Hamburg, providing a research environment centered on structural biology and native mass spectrometry. The non-academic placement at Bruker Daltonics connected the academic research directly to mass spectrometry instrument development, enabling knowledge transfer in both directions between fundamental research and industrial application.
The broader objective was to establish a methodology applicable beyond AMPK, contributing to the molecular characterization of disease-relevant kinase complexes and illustrating how fundamental research in structural proteomics can inform future clinical development.
A recombinant AMPK protein platform was established as the foundation for the project, enabling the development of a hybrid native top-down mass spectrometry methodology for kinase complex characterization. This led to detailed investigation of AMPK proteoforms, phosphorylation dynamics, and nucleotide binding heterogeneity, resulting in two publications directly reporting AMPK research, with the methodology additionally contributing to three further collaborative peer-reviewed publications in adjacent fields. The methodology is transferable beyond AMPK to other kinase complexes, and has already been applied in collaborative studies on coronavirus polyprotein processing and gas-phase protein complex characterization. A related nanoparticle enrichment project initiated during the fellowship is ongoing at UW-Madison, and the analytical methodology has been extended to acetyl-CoA carboxylase as a second major metabolic target through a supervised master thesis project.
The return phase at CSSB Hamburg and University of Lübeck focused on consolidating and publishing results, strengthening the research line, and developing industry collaborations. A collaboration with Bruker Daltonics supported the development of new applications and data analysis pipelines, with mutual benefit: the researcher gained hands-on expertise in advanced instrumentation, while Bruker could benchmark systems with complex protein targets not typically accessible in an industry setting.
Knowledge transfer was an integral part of the project, with four PhD and master students directly involved. The AMPK research platform served as a basis for their individual research projects, contributing to a next generation of researchers trained in hybrid top-down mass spectrometry approaches and in several cases leading to independent publications.
The return phase at CSSB Hamburg and University of Lübeck focused on consolidating and publishing results, strengthening the research line, and developing industry collaborations. A collaboration with Bruker Daltonics supported the development of new applications and data analysis pipelines, with mutual benefit: the researcher gained hands-on expertise in advanced instrumentation, while Bruker could benchmark systems with complex protein targets not typically accessible in an industry setting.
Knowledge transfer was an integral part of the project, with four PhD and master students directly involved. The AMPK research platform served as a basis for their individual research projects, contributing to a next generation of researchers trained in hybrid top-down mass spectrometry approaches and in several cases leading to independent publications.
Conventional mass spectrometry approaches typically analyze proteins as averaged populations, losing the detail of co-existing proteoforms and their individual modification states. By applying native top-down and top-down mass spectrometry in a hybrid framework, this project resolved the structural heterogeneity and proteoform landscape of AMPK at a level of detail not previously accessible. Non-covalent interactions and phosphorylation states were characterized simultaneously while preserving complex integrity, revealing proteoform-based regulatory mechanisms and new insights into how chemical heterogeneity at the molecular level relates to the functional regulation of this metabolic kinase.
Further integration with computational biology approaches could help translate these molecular insights toward clinical applications. Of particular interest is understanding how the identified phosphorylation states and proteoform distributions are established and maintained in the cellular context, which would require collaborative efforts spanning mass spectrometry, cell biology, and clinical research.
Further integration with computational biology approaches could help translate these molecular insights toward clinical applications. Of particular interest is understanding how the identified phosphorylation states and proteoform distributions are established and maintained in the cellular context, which would require collaborative efforts spanning mass spectrometry, cell biology, and clinical research.