Mass spectrometry: a window into native protein structure, conformation, dynamic, and interactions

The present dissertation addresses the theoretical foundations, methodological development, and practical application of chromatography, ion mobility spectrometry, and mass spectrometry in the field of protein and peptide analysis. The chromatographic section focuses on the definition and technical principles of ultra-high-performance liquid chromatography (UHPLC) and nano-liquid chromatography, reversed-phase chromatography, size-exclusion chromatography (SEC), as well as online solid-phase extraction and online protein digestion. Within the field of ion mobility spectrometry (IMS), the fundamental definition of the technique is presented, together with the operating principles of drift-tube ion mobility spectrometry (DTIMS) and travelling-wave ion mobility spectrometry (TWIMS). Influencing factors, such as the selection of the buffer gas, are discussed, along with the determination of collision cross sections (CCS) using both absolute and relative analytical approaches. In addition, computational (in silico) methods for calculating collision cross sections from protein structures are described. Mass spectrometry served as the detection system throughout this work. Accordingly, the fundamental architecture of a mass spectrometer is outlined, with particular emphasis on key components such as ion optics and detectors. Among the mass analyzers, the operating principles of the low-resolution quadrupole and Paul ion trap, as well as the high-resolution time-of-flight (TOF) analyzer and the Orbitrap analyzer, are explained. The electrospray ionization (ESI) source, which is of central importance for protein analysis and for this dissertation, is described in detail, and the inherent incompatibilities between liquid chromatography and mass spectrometry are critically discussed. Protein analysis is introduced by a chapter covering proteins in general, including their structural hierarchy, biological functions, and the structural organization of antibodies. The application of the aforementioned analytical techniques in protein analysis is subsequently presented. Examples include the measurement of intact proteins with a mass accuracy below 10 ppm, peptide mapping, the detection of phosphorylated peptides using multidimensional protein identification technology (MudPIT), epitope and paratope mapping by footprinting techniques such as hydrogen–deuterium exchange mass spectrometry (HDX-MS), triple confirmation of protein folding by SEC–IM–MS, imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the differentiation of IgG1, IgG2, IgG3, and IgG4 subclasses using IM-MS. The research results include the development of a novel oxidative footprinting method, which was validated and optimized using exemplary proteins such as FKBP51. FKBP51 occupies a central position in a parallel research project aimed at enhancing its activity. In this context, IM-MS, SEC, electron transfer dissociation (ETD), and intact mass analysis were employed to demonstrate crosslinking via lactam bridges in fully synthetic FKBP variants containing noncanonical amino acids, as well as disulfide bond formation in recombinant FKBP51. Using IM-MS in combination with calculated CCS values, studies on HDAC4 demonstrated that crystal structures do not always accurately represent the dynamic conformational ensemble of proteins in solution, and that techniques such as IM-MS are better suited to capture these structural dynamics. For Dicer, a key protein involved in mRNA degradation and translational repression, hydrogen–deuterium exchange mass spectrometry (HDX-MS) revealed a previously unrecognized interaction with 5-lipoxygenase (5-LO), a protein associated with inflammatory responses. In summary, this dissertation provides a comprehensive introduction to chromatography, mass spectrometry, ion mobility spectrometry, proteins, and protein structure. At the same time, these techniques are applied in practical research projects that demonstrate their advantages and versatility in modern biochemical research.