Comparing Solution-Phase and Gas-Phase Protein Stability Using Ion Mobility and Differential Mobility Mass Spectrometry

A critical quality attribute for many proteins concerning biopharmaceutical products is the proteins stability. Current stability assays are lengthy but some quick stability assays to determine the proteins melting temperature include thermal ramps of a protein using a fluorescent dye to monitor unfolding. Protein unfolding has also been studies with mass spectrometry through collision induced unfolding (CIU) whereby the ion temperature in the trap collision cell is increased and unfolding changes are tracked in the ion mobility cell. However, whether a protein retains its structural properties in the gas phase has been a topic of wide discussion. Here, we present that CIU and solution thermal melts provide the same information on protein stability using a homo-tetrameric model protein, pyruvate kinase, and four of its point mutants. Efforts to employ a second ion mobility technique differential mobility spectrometry, to determine gas phase stability will also be discussed

Imaging mass spectrometry approaches for the detection and localisation of drug compounds and small molecules in tissue

A crucial and challenging aspect of the drug development process is the requirement to measure the distribution of a pharmaceutical compound and its metabolites in tissue. Industry-standard methods used to look at total localisation of drug-related material are limited due to their dependence on labels. These labelled techniques can have difficulty in distinguishing between the drug of interest and its metabolites. Imaging mass spectrometry is a technique that has the potential to spatially distinguish between drug and metabolites, due to its high chemical specificity and sensitivity. A number of imaging mass spectrometry approaches have been described for localisation of drug compounds in tissue, most notably matrix-assisted laser desorption/ionisation (MALDI) imaging, which can provide data complementary to existing imaging techniques. Two imaging mass spectrometry approaches have been evaluated and compared for use in the localisation of a range of drug compounds in target tissues. The techniques used were MALDI imaging and a recently described electrospray ionisation-based technique, liquid extraction surface analysis (LESA). Both techniques have been successfully used for the detection of drug compounds in dosed tissue sections. A major challenge associated with imaging techniques is the required selectivity of the experiment for the compound of interest, due to the complex nature of tissue sections. Combining the shape-selective method of ion mobility separation with MS/MS fragmentation has been shown to improve the selectivity of both imaging approaches for the compound of interest. Results obtained using LESA-MS have demonstrated the suitability of this technique as a rapid and sensitive profiling technique for the detection of drugs and metabolites in tissue, but with a lower achievable spatial resolution than MALDI imaging. Higher spatial resolution was achieved with MALDI imaging; however data acquisition times were longer and required higher dosing levels for successful detection of drug compounds in tissue. A biological application of MALDI imaging was also evaluated. Mobility-enabled MALDI imaging was used to assess differences in the localisation of important adenine nucleotides between control and metabolically stressed mouse brain sections. Tissue fixation methods were evaluated to overcome rapid post-mortem degradation of adenine nucleotides such that biologically relevant localisation images can be obtained. These studies highlight the crucial importance of appropriate biological sample preparation in MALDI imaging experiments

Study of π0\pi^0 pair production in single-tag two-photon collisions

We report a measurement of the differential cross section of $\pi^0$ pair production in single-tag two-photon collisions, $\gamma^* \gamma \to \pi^0 \pi^0$, in $e^+ e^-$ scattering. The cross section is measured for $Q^2$ up to 30 GeV$^2$, where $Q^2$ is the negative of the invariant mass squared of the tagged photon, in the kinematic range 0.5 GeV < W < 2.1 GeV and $|\cos \theta^*|$ < 1.0 for the total energy and pion scattering angle, respectively, in the $\gamma^* \gamma$ center-of-mass system. The results are based on a data sample of 759 fb$^{-1}$ collected with the Belle detector at the KEKB asymmetric-energy $e^+ e^-$ collider. The transition form factor of the $f_0(980)$ and that of the $f_2(1270)$ with the helicity-0, -1, and -2 components separately are measured for the first time and are compared with theoretical calculations.Comment: 36 pages, 37 figures, 11 tables, Belle Preprint 2015-15, KEK Preprint 2015-2

Applications of ion mobility spectrometry, collision-induced dissociation and electron activated dissociation tandem mass spectrometry to structural analysis of proteins, glycoproteins and glycans

This dissertation mainly focuses on analytical method development for characterization of proteins, glycoproteins and glycans using the recently developed ion mobility spectrometry (IMS) techniques and various electron activated dissociation (ExD) tandem mass spectrometry methods. IMS and ExD have become important techniques in structure analysis of biomolecules. IMS is a gas-phase separation method orthogonal to liquid chromatography (LC) fractionation. ExD is capable of producing a large number of structurally informative fragment ions for elucidation of structural details, complementary to collision-induced dissociation (CID). We first applied the selected accumulation-trapped IMS (SA-TIMS)-electronic excitation dissociation (EED) method to analyze various mixtures of glycan isomers. Glycan linkage isomers with linear or branched structure were successfully separated and subsequently identified. Theoretical modeling was also performed to gain a better understanding of isomer separation. The calculated collisional cross section (CCS) values match well with the experimentally measured ones, and suggested that the choice of metal charge carrier and charge state is critical for successful IMS separation of isomeric glycans. In addition, a SA-TIMS-electron capture dissociation (ECD) approach was employed to study gas-phase protein conformation, as the ECD fragmentation pattern is influenced by both the charge distribution and the presence of various non-covalent interactions. We demonstrated that different conformations of protein ions in a single charge state could produce distinct fragmentation pattern, presumably because of their differences in tertiary structures and/or proton locations. The second part describes characterization of glycoproteins using LC-hot ECD. To improve the cleavage coverage of glycopeptides, hot ECD, a fragmentation method utilizing the irradiation of high-energy electrons, was optimized for both middle-down and bottom-up analyses of glycopeptides, including peptides with multiple glycosylation sites. Hot ECD was shown to be an effective fragmentation technique for sequencing of glycopeptides, even for ions in lower charge states. In addition, the online LC-hot ECD approach was applied to characterize extensively modified glycoproteins from biological sources in which all glycosylation sites could be unambiguously determined. This study expands the applications of IMS, CID and ExD to structural analysis of various biomolecules, and explores the analytical potential of combining them for investigation of complex biological systems, in particular, enzyme mechanisms

A Novel Approach To Intelligent Navigation Of A Mobile Robot In A Dynamic And Cluttered Indoor Environment

The need and rationale for improved solutions to indoor robot navigation is increasingly driven by the influx of domestic and industrial mobile robots into the market. This research has developed and implemented a novel navigation technique for a mobile robot operating in a cluttered and dynamic indoor environment. It divides the indoor navigation problem into three distinct but interrelated parts, namely, localization, mapping and path planning. The localization part has been addressed using dead-reckoning (odometry). A least squares numerical approach has been used to calibrate the odometer parameters to minimize the effect of systematic errors on the performance, and an intermittent resetting technique, which employs RFID tags placed at known locations in the indoor environment in conjunction with door-markers, has been developed and implemented to mitigate the errors remaining after the calibration. A mapping technique that employs a laser measurement sensor as the main exteroceptive sensor has been developed and implemented for building a binary occupancy grid map of the environment. A-r-Star pathfinder, a new path planning algorithm that is capable of high performance both in cluttered and sparse environments, has been developed and implemented. Its properties, challenges, and solutions to those challenges have also been highlighted in this research. An incremental version of the A-r-Star has been developed to handle dynamic environments. Simulation experiments highlighting properties and performance of the individual components have been developed and executed using MATLAB. A prototype world has been built using the WebotsTM robotic prototyping and 3-D simulation software. An integrated version of the system comprising the localization, mapping and path planning techniques has been executed in this prototype workspace to produce validation results