Higher-order-structure analysis of proteins by native size-based separations coupled to optical and mass-spectrometric detectors

Continuous developments focussed on ever larger and more-complex molecules, including their supramolecular assemblies, have opened new frontiers and opportunities in many domains of chemistry and, especially, in life sciences, medicine, and biotechnology. Biopharmaceuticals and biotechnological proteins are among the most sophisticated and elegant products of modern science. These highly complex structures possess advanced properties and improved functions, which are directly related to their higher-order structure (HOS) and chemical composition. To develop such products and to ensure their efficacy and safety, suitable analytical methods are crucial. Preservation of the structural integrity of the HOS and the functional native form during analysis is an absolute prerequisite to draw meaningful and reliable conclusions, especially in the characterization of labile macromolecules. In this project the objective was to establish novel analytical platforms by combining advanced separation techniques with state-of-the-art mass spectrometry. The developed platforms provide detailed information on the size, chemical composition and conformation of labile (bio)macromolecules and their HOS in a single analysis. We have performed extensive investigations into the suitability of the size-based separation techniques, mainly size-exclusion chromatography and asymmetrical flow field-flow fractionation, for the non-destructive characterization of proteins under (near-) native conditions. Emphasis has been on optimization of the separation and interfacing with several detectors that provide complementary structural and chemical information. The latter include liquid-phase characterization by multi-angle light scattering (MALS), UV absorbance, and refractive-index detection, and gas-phase detection by high-resolution mass spectrometry. Ultimately, these synergistic platforms were proven pivotal to understand possible structural alterations during the analysis and to ensure conditions that can provide a reliable picture of the actual (native) state of proteins and labile protein complexes in solution