Probing Structure and Stability of Biomolecules with Ion Mobility-mass Spectrometry Techniques

Abstract

Ion mobility spectrometry – mass spectrometry (IMS-MS) based techniques have significantly advanced the study of both the structure and stability of biomolecules. Solution phase, biologically relevant peptide/protein conformations have been analyzed utilizing electrospray ionization (ESI) coupled to IMS-MS. The work presented here focuses on the development and application of IMS-MS for the study of the structure and stability of peptides and proteins. Structures originating in solution can be related to the biological activity of the system. Specifically, we examine the influence of both proline isomerization and metal binding on the conformational preferences and stabilities of a series of peptides and proteins.

The first study uses IMS-MS to examine the conformation specific binding of Zn²⁺ to oxytocin (OT). When bound to Zn, OT has an increased affinity for its receptor. We observe two conformations which are the result of the cis-trans orientation of the Cys⁶-Pro ⁷ bond. We find that Zn²⁺ binds preferentially to the trans configuration demonstrating that cis-trans isomerization regulates the binding of Zn²⁺ to OT, therefore linking the orientation of the Cys⁶-Pro⁷ bond to the biological activity of the molecule. In another series of experiments, we look at the role of penultimate proline on the stability of bradykinin along with a library of penultimate proline containing peptides. For BK, we find that the Pro²-Pro³ bond, which is enzymatically resistant, is cleaved with 100% specificity when incubated at high temperatures. With IMS-MS, we are able to monitor the mass spectral and conformational changes as a function of time, and find that cis-trans isomerization of a single bond regulates the rate of dissociation, and hence the stability of the system. Finally, we look at the thermal stability of the insulin-zinc hexamer complex. Utilizing temperature-controlled nano-ESI IMS-MS, we capture the dissociation of the insulin hexamer and the unfolding of insulin dimer and monomer, occurring simultaneously with increasing solution temperature. We show the ability of IMS-MS to observe the conformation specific melting temperatures of a protein complex, demonstrating a fast and sensitive assay for stability characterization.