Investigating protein structure by means of mass spectrometry

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

Investigating protein structure by means of mass spectrometry

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.EThOS - Electronic Theses Online ServiceBiotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)Waters CorporationGBUnited Kingdo

Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7 angstrom range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.Peer reviewe

Examination of ataxin-3 (atx-3) aggregation by structural mass spectrometry techniques: A rationale for expedited aggregation upon polyglutamine (polyQ) expansion

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73-96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided

Characterization of complex polysorbate formulations by means of shape-selective mass spectrometry

Complex synthetic formulations based on polysorbates can be challenging to characterize. They may be composed of many similar products including those of the same molecular weight, which cannot be readily separated by separation science approaches. Carbon number variation and ethylene oxide distribution add to the complexity. The properties of these formulations will be dependent on the chemical structure and relative concentration of formulation components. Here we describe the use of two experimental approaches based on mass spectrometry to provide enhanced characterization of these formulations. The first utilizes an atmospheric pressure solids analysis probe to rapidly determine the percentage content of individual esters in a formulation. These are shown to be in good agreement with product specification sheets. In a second approach, mobility separation has been integrated into a MALDI-MS/MS experiment to categorize major, minor, and trace ingredients. Components of identical molecular mass in the polysorbate formulations have been separated by ion mobility and then fragmented for additional characterization. The rapidity and level of structural detail provided by these experiments offers a significant opportunity to develop practical screening methods for complex formulations

Travelling wave ion mobility mass spectrometry studies of protein structure : biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements

The three-dimensional conformation of a protein is central to its biological function. The characterisation of aspects of three-dimensional protein structure by mass spectrometry is an area of much interest as the gas-phase conformation, in many instances, can be related to that of the solution phase. Travelling wave ion mobility mass spectrometry (TWIMS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIMS under denaturing and near-physiological solvent conditions and cross-sections estimated for the charge states observed. Estimates of collision cross-sections were obtained with reference to known standards with published cross-sections. Estimated cross-sections were compared with values from published X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy structures. The cross-section measured by ion mobility mass spectrometry varies with charge state, allowing the unfolding transition of proteins in the gas phase to be studied. Cross-sections estimated experimentally for proteins studied, for charge states most indicative of native structure, are in good agreement with measurements calculated from published X-ray and NMR structures. The relative stability of gas-phase structures has been investigated, for the proteins studied, based on their change in cross-section with increase in charge. These results illustrate that the TWIMS approach can provide data on three-dimensional protein structures of biological relevance. Copyright (C) 2008 John Wiley & Sons, Ltd

Meticillin-resistant Staphylococcus aureus (MRSA) in hospitals and the community: model predictions based on the UK situation

Theoretical modelling has shown that patient movements in and out of hospitals are likely to affect nosocomial transmission dynamics considerably. The community acts as a "reservoir" and readmission of individuals colonised during previous admissions can result in sporadic transmission episodes within hospitals. We investigated patient movement patterns and frequency of readmissions using seven years of complete data from the University Hospitals of Leicester NHS Trust. Sufficient information is held on individual patients to study the heterogeneity in readmission. Overall, we found that an infected person has a 44.2% chance of being readmitted to the Trust white still infected. This value is far higher than previous estimates (3.7% [Cooper et al., Health Technol Assess 2003;7(39)]), highlighting the potential importance of transmission driven by hospital admissions. For this reason we believe consideration of readmissions from the community population to be critical to the success of hospital acquired infection control. (C) 2007 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved

Characterization of Complex Polysorbate Formulations by Means of Shape-Selective Mass Spectrometry

Complex synthetic formulations based on polysorbates can be challenging to characterize. They may be composed of many similar products including those of the same molecular weight, which cannot be readily separated by separation science approaches. Carbon number variation and ethylene oxide distribution add to the complexity. The properties of these formulations will be dependent on the chemical structure and relative concentration of formulation components. Here we describe the use of two experimental approaches based on mass spectrometry to provide enhanced characterization of these formulations. The first utilizes an atmospheric pressure solids analysis probe to rapidly determine the percentage content of individual esters in a formulation. These are shown to be in good agreement with product specification sheets. In a second approach, mobility separation has been integrated into a MALDI-MS/MS experiment to categorize major, minor, and trace ingredients. Components of identical molecular mass in the polysorbate formulations have been separated by ion mobility and then fragmented for additional characterization. The rapidity and level of structural detail provided by these experiments offers a significant opportunity to develop practical screening methods for complex formulations

Resolution of a paradox by native mass spectrometry : facile occupation of all four metal binding sites in the dimeric zinc sensor SmtB

The predominant species of the cyanobacterial metalloregulatory protein SmtB as observed by ESI-MS is a dimer with all four zinc binding sites occupied

Estimating Collision Cross Sections of Negatively Charged N-Glycans using Traveling Wave Ion Mobility-Mass Spectrometry

Glycosylation is one of the most common post-translational modifications occurring in proteins. A detailed structural characterization of the involved carbohydrates, however, is still one of the greatest challenges in modern glycoproteomics, since multiple regio- and stereoisomers with an identical monosaccharide composition may exist. Recently, ion mobility-mass spectrometry (IM-MS), a technique in which ions are separated according to their mass, charge, and shape, has evolved as a promising technique for the separation and structural analysis of complex carbohydrates. This growing interest is based on the fact that the measured drift times can be converted into collision cross sections (CCSs), which can be compared, implemented into databases, and used as additional search criteria for structural identification. However, most of the currently used commercial IM-MS instruments utilize a nonuniform traveling wave field to propel the ions through the IM cell. As a result, CCS measurements cannot be performed directly and require calibration. Here, we present a calibration data set consisting of over 500 reference CCSs for negatively charged N-glycans and their fragments. Moreover, we show that dextran, already widely used as a calibrant in high performance liquid chromatography, is also a suitable calibrant for CCS estimations. Our data also indicate that a considerably increased error has to be taken into account when reference CCSs acquired in a different drift gas are used for calibration