Investigating protein structure by means of mass spectrometry

Abstract

The three-dimensional conformation of a protein is central to its biological function. Mass spectrometry (MS) has become an important tool for the study of various aspects of protein structure. This project investigates the use of MS for diagnosis of hemoglobinopathies, through primary structure identification, and for threedimensional protein structure analysis, through comparison to established methods and application to protein systems. Travelling-wave ion mobility mass spectrometry (TWIM-MS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIM-MS. Cross-sections were estimated for proteins studied, for charge states most indicative of native structure, and were found to be in good agreement with those calculated from published X-ray crystallography and nuclear magnetic resonance structures. These results illustrated that the TWIM-MS approach can provide biologically-relevant data on three-dimensional protein structure. TWIM-MS was then used to study the structural properties of the hemoglobin tetramer and its components. Results showed that globin monomers exist in similar conformations whether in apo- or holo- forms and that a heme-deficient dimer is unlikely to be a prerequisite for hemoglobin tetramer assembly. TWIM-MS was used to successfully differentiate between normal and sickle hemoglobin tetramers. The conformational changes occurring in VanS, a histidine kinase, upon autophosphorylation were investigated by TWIM-MS. Results provided insights into the mechanism of autophosphorylation. MS was used to follow the rate of the autophosphorylation and results obtained compared well with those from an established method. This demonstrated that MS offers a simple, reproducible alternative to conventional methods for the study of phosphorylation rates. MS was used to provide positive identification of a range of hemoglobinopathies caused by single point mutations. A high-throughput method was used to screen for hemoglobinopathies in South Asians with and without cardiovascular disease. Results showed a positive correlation between patients with hemoglobinopathies and those with cardiovascular disease