Development of Mass Spectrometry-based Workflows to Study Metal Interactions and Dynamics in the Extracellular Space

The works included in this dissertation focus on the application of analytical and biochemical methodologies to study metal-binding and metal-regulating biomolecules. Additionally, aspects of molecular biology, programming, and spectroscopy are found in these works. This thesis describes the implementation of mass spectrometry-based techniques to understand metal dynamics in the extracellular space. Chapter One describes the current state of mass-spectrometry-based applications toward the discovery of metal-binding species and methods to determine metal-interaction sites. This chapter highlights techniques for understanding metal dynamics of metal-binding biomolecules as well as monitoring the distribution of metal micronutrient speciation in the extracellular space. Topics include sample preparation, native and non-native mass spectrometry (NMS), size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS), and immobilized metal affinity chromatography (IMAC). This chapter will provide the framework for the following chapters in this thesis. Chapter Two describes efforts to elucidate the zinc and copper binding propensity and binding site of the peptide hormone C-peptide. Methods used include mass spectrometry (MS), nuclear magnetic resonance (NMR), ultraviolet–visible light spectroscopy, and isothermal calorimetry (ITC). These studies determined that Cu2+ binding occurs primarily at the N-terminus; however, other interaction sites beyond what could be confidently detected may occur at the C-terminal region. Chapter Three focuses on identifying copper-binding peptides by IMAC. This chapter includes work towards the development and the utilization of IMAC to enrich synthetic metal binding peptides as well as the peptide hormone hepcidin – showing possible structural dependence of Hepcidin for Cu2+ mediated ternary complex formation. IMAC work was further extended to identify Cu2+ binding peptides from enzymatically digested rice bran. Specific peptides were selected based ...