Giants in Orbit: Orbitrap based native MS studies on large biomolecules

Abstract

This thesis illustrates challenges, opportunities, and solutions we encounter in the analysis of high mass samples by native MS. These samples can exhibit extraordinary heterogeneity caused by either a variable stoichiometry or extensive glycosylation. The challenge we face in the analysis of such analytes is the need to resolve charge states for the charge assignment, which is based on the relative positioning of ion signals. This can be substituted by an independent measure of charge as employed in CDMS. In Chapter 2, we demonstrate single particle CDMS on the Orbitrap, introducing a second dimension to resolve coinciding ions and the ability to predict their mass individually with an RMSD of about 3.5 charges at transient lengths of ~1s. The ability to measure ions individually offers the opportunity to get a better understanding of their behavior in the mass spectrometer compared to only observing their convoluted ion signals. This was done in Chapter 3, where we tracked individual ions through their lifetime in the Orbitrap mass analyzer and discovered a surprisingly high stability for large ions compared to smaller analytes. Besides that, we could observe small frequency drifts caused by solvent losses in the Orbitrap, which allowed us to develop several strategies for improved CDMS. In the following Chapters 4-6, we present experimental solutions for the characterization of increasingly heterogeneous assemblies, complemented with exciting insights into their biology. In Chapters 4, we investigate the cooperativity of the assembly reaction for designed nanomaterials using tandem MS for an unambiguous identification of complete assemblies with exactly 120 subunits. In Chapter 5, we analyze recombinant AAV, which can occur in 1891 different stoichiometries, attributed to their variable composition of 60 copies of any of its three capsids proteins. We approached this by simulating all possible ions and compared their convoluted signals with the experimental data, thereby verifying a stochastic assembly mechanism. In Chapter 6, we analyzed genome loaded AAV, which have an even greater degree of heterogeneity caused by the variable size of their genomes. Such an extreme degree of heterogeneity can still be analyzed by CDMS and we present a sensitive approach for the identification and quantification of empty and filled recombinant AAV.