Gas Phase Stability and Unfolding of Proteins via Ion Mobility-Mass Spectrometry

The study of protein dynamics and unfolding is key to understanding the basis of misfolding and aggregational diseases such as type II diabetes, α1-antrypsin deficiency, Parkinsons’ disease and Alzheimers' disease. Common structural biology techniques, such as x-ray crystallography and cryo electron microscopy often require large quantities of high purity sample and can only capture major structural ensembles. Mass spectrometry coupled to ion mobility (IM-MS) is positioned as an ideal technique to study protein dynamics and unfolding as it allows the separation of ions based on mass, charge and conformation from low concentrations of heterogenous solutions. In this thesis I present new techniques, instrumentation and computational workflows relating to IM-MS and how these techniques can be applied to protein systems involved in misfolding and aggregational diseases

Linking Gas-Phase and Solution-Phase Protein Unfolding via Mobile Proton Simulations

Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase (in vacuo) is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between in vacuo and in-solution behavior, we use unbiased molecular dynamics (MD) to create in silico models of in vacuo unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded in silico models for observed charge states of NTL9. The unfolding behavior in silico replicates the behavior in-solution and is in line with the in vacuo observations; however, the theoretical collision cross section (CCS) of the in silico models was lower compared to that of the in vacuo data, which may reflect reduced sampling

Developments in tandem ion mobility mass spectrometry

Ion Mobility (IM) coupled to mass spectrometry (MS) is a useful tool for separating species of interest out of small quantities of heterogenous mixtures via a combination of m/z and molecular shape. While tandem MS instruments are common, instruments which employ tandem IM are less so with the first commercial IM–MS instrument capable of multiple IM selection rounds being released in 2019. Here we explore the history of tandem IM instruments, recent developments, the applications to biological systems and expected future directions

Concentration‐dependent coulombic effects in travelling wave ion mobility spectrometry collision cross section calibration

RationaleTravelling wave ion mobility spectrometry (TWIMS) is increasingly being used as a method for calculating the collision cross sections (CCSs) of protein ions. To calculate the CCS values of unknown ions, however, the TWIMS device needs to be calibrated using calibrant proteins of known CCS values. The effect of calibrant protein concentration on the accuracy of the resulting calibration curve has not been explicitly studied so far. We hypothesised that at high protein concentrations the ion density within the TWIMS device will be such that ions will experience space charge effects resulting in deviations, as well as broadening, of ion arrival time distributions (ATDs). Calibration curves using these altered ATDs would therefore result in incorrect CCS values being calculated for the protein ions of interest.MethodsThree protein CCS calibrants, avidin, bovine serum albumin and β‐lactgobulin, were prepared at different concentrations and used to calculate the CCS of a non‐calibrant protein. Data were collected on a Synapt G1 ion mobility mass spectrometer with a nano‐electrospray ionisation (nESI) source using capillaries prepared in house.ResultsIncreasing the concentration of CCS calibrants caused ATD broadening and shifted the ATD peak tops, leading to a significant increase in calculated CCS values.ConclusionsThe concentration of protein calibrants can directly affect the quality of the CCS calibration in TWIMS experiments

Suppression of O-linked glycosylation of the SARS-CoV-2 spike by quaternary structural restraints

Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.</p

Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Travelling Wave Device

Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into “conformational families”. Increasing the IM resolving power should allow finer structural information to be obtained, and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds. Here, we report on investigations of protein ion stability using a multi-pass travelling wave (TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A). The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed which were used to obtain more detailed collision induced unfolding pathways for Cytochrome C. The novel instrument geometry provide unique capabilities with the potential to expand the field of protein analysis via IM-MS.</div

Analysis of Proline Substitutions Reveals the Plasticity and Sequence Sensitivity of Human IAPP Amyloidogenicity and Toxicity

Pancreatic amyloid formation by the polypeptide IAPP contributes to β-cell dysfunction in type 2 diabetes. There is a 1:1 correspondence between the ability of IAPP from different species to form amyloid in vitro and the susceptibility of the organism to develop diabetes. Rat IAPP is non-amyloidogenic and differs from human IAPP at six positions, including three proline replacements: A25P, S28P, and S29P. Incorporation of these proline residues into human IAPP leads to a non-amyloidogenic analogue that is used clinically. The role of the individual proline residues is not understood. We examine the three single and three double proline substitutions in the context of human IAPP. An S28P substitution significantly decreases amyloidogenicity and toxicity, while an S29P substitution has very modest effects despite being an identical replacement just one residue away. The consequences of the A25P substitution are between those of the two Ser to Pro substitutions. Double analogues containing an S28P replacement are less amyloidogenic and less toxic than the IAPPA25P&nbsp;S29P double analogue. Ion mobility mass spectrometry reveals that there is no correlation between the monomer or dimer conformation as reported by collision cross section measurements and the time to form amyloid. The work reveals both the plasticity of IAPP amyloid formation and the exquisite sequence sensitivity of IAPP amyloidogenicity and toxicity. The study highlights the key role of the S28P substitution and provides information that will aid in the rational design of soluble variants of IAPP. The variants studied here offer a system for further exploring features that control IAPP toxicity

VEGFA, B, C: Implications of the C-terminal sequence variations for the interaction with neuropilins

Vascular endothelial growth factors (VEGFs) are the key regulators of blood and lymphatic vessels’ formation and function. Each of the proteins from the homologous family VEGFA, VEGFB, VEGFC and VEGFD employs a core cysteine-knot structural domain for the specific interaction with one or more of the cognate tyrosine kinase receptors. Additional diversity is exhibited by the involvement of neuropilins–transmembrane co-receptors, whose b1 domain contains the binding site for the C-terminal sequence of VEGFs. Although all relevant isoforms of VEGFs that interact with neuropilins contain the required C-terminal Arg residue, there is selectivity of neuropilins and VEGF receptors for the VEGF proteins, which is reflected in the physiological roles that they mediate. To decipher the contribution made by the C-terminal sequences of the individual VEGF proteins to that functional differentiation, we determined structures of molecular complexes of neuropilins and VEGF-derived peptides and examined binding interactions for all neuropilin-VEGF pairs experimentally and computationally. While X-ray crystal structures and ligand-binding experiments highlighted similarities between the ligands, the molecular dynamics simulations uncovered conformational preferences of VEGF-derived peptides beyond the C-terminal arginine that contribute to the ligand selectivity of neuropilins. The implications for the design of the selective antagonists of neuropilins’ functions are discussed

Analysis of proline substitutions reveals the plasticity and sequence sensitivity of human IAPP amyloidogenicity and toxicity

Pancreatic amyloid formation by the polypeptide IAPP contributes to β-cell dysfunction in type 2 diabetes. There is a 1:1 correspondence between the ability of IAPP from different species to form amyloid in vitro and the susceptibility of the organism to develop diabetes. Rat IAPP is non-amyloidogenic and differs from human IAPP at six positions, including three proline replacements: A25P, S28P, and S29P. Incorporation of these proline residues into human IAPP leads to a non-amyloidogenic analogue that is used clinically. The role of the individual proline residues is not understood. We examine the three single and three double proline substitutions in the context of human IAPP. An S28P substitution significantly decreases amyloidogenicity and toxicity, while an S29P substitution has very modest effects despite being an identical replacement just one residue away. The consequences of the A25P substitution are between those of the two Ser to Pro substitutions. Double analogues containing an S28P replacement are less amyloidogenic and less toxic than the IAPPA25P S29P double analogue. Ion mobility mass spectrometry reveals that there is no correlation between the monomer or dimer conformation as reported by collision cross section measurements and the time to form amyloid. The work reveals both the plasticity of IAPP amyloid formation and the exquisite sequence sensitivity of IAPP amyloidogenicity and toxicity. The study highlights the key role of the S28P substitution and provides information that will aid in the rational design of soluble variants of IAPP. The variants studied here offer a system for further exploring features that control IAPP toxicity

Analysis of Baboon IAPP provides insight into amyloidogenicity and cytotoxicity of human IAPP

The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic β-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured β-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured β-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation