IU Indianapolis ScholarWorks :: Browsing by Author "Webb, Ian K."

Browsing by Author "Webb, Ian K."

Now showing 1 - 10 of 12

Advances in Gas Chromatography and Vacuum UV Spectroscopy: Applications to Fire Debris Analysis & Drugs of Abuse
(2020-12) Roberson, Zackery Ray; Goodpaster, John V.; Manicke, Nicholas E.; Webb, Ian K.; Laulhé, Sébastien
In forensic chemistry, a quicker and more accurate analysis of a sample is always being pursued. Speedy analyses allow the analyst to provide quick turn-around times and potentially decrease back-logs that are known to be a problem in the field. Accurate analyses are paramount with the futures and lives of the accused potentially on the line. One of the most common methods of analysis in forensic chemistry laboratories is gas chromatography, chosen for the relative speed and efficiency afforded by this method. Two major routes were attempted to further improve on gas chromatography applications in forensic chemistry. The first route was to decrease separation times for analysis of ignitable liquid residues by using micro-bore wall coated open-tubular columns. Micro-bore columns are much shorter and have higher separation efficiencies than the standard columns used in forensic chemistry, allowing for faster analysis times while maintaining the expected peak separation. Typical separation times for fire debris samples are between thirty minutes and one hour, the micro-bore columns were able to achieve equivalent performance in three minutes. The reduction in analysis time was demonstrated by analysis of ignitable liquid residues from simulated fire debris exemplars. The second route looked at a relatively new detector for gas chromatography known as a vacuum ultraviolet (VUV) spectrophotometer. The VUV detector uses traditional UV and far-ultraviolet light to probe the pi and sigma bonds of the gas phase analytes as well as Rydberg traditions to produce spectra that are nearly unique to a compound. Thus far, the only spectra that were not discernable were from enantiomers, otherwise even diastereomers have been differentiated. The specificity attained with the VUV detector has achieved differentiation of compounds that mass spectrometry, the most common detection method for chromatography in forensic chemistry labs, has difficulty distinguishing. This specificity has been demonstrated herein by analyzing various classes of drugs of abuse and applicability to “real world” samples has been demonstrated by analysis of de-identified seized samples.
Application of Multiple Length Crosslinkers to the Characterization of Gaseous Protein Structure
(American Chemical Society, 2022-09-13) Kit, Melanie Cheung See; Webb, Ian K.; Chemistry and Chemical Biology, School of Science
The speed, sensitivity, and tolerance of heterogeneity of native mass spectrometry, as well as the kinetic trapping of solution-like states during electrospray, makes mass spectrometry an attractive method to study protein structure. Increasing resolution of ion mobility measurements and mass resolving power and range are leading to the increase of the information content of intact protein measurements, and an expanded role of mass spectrometry in structural biology. Herein, a suite of different length noncovalent (sulfonate to positively charged side chain) crosslinkers was introduced via gas-phase ion/ion chemistry and used to determine distance restraints of kinetically trapped gas-phase structures of native-like cytochrome c ions. Electron capture dissociation allowed for the identification of crosslinked sites. Different length linkers resulted in distinct pairs of side chains being linked, supporting the ability of gas-phase crosslinking to be structurally specific. The gas-phase lengths of the crosslinkers were determined by conformational searches and density functional theory, allowing for the interpretation of the crosslinks as distance restraints. These distance restraints were used to model gas-phase structures with molecular dynamics simulations, revealing a mixture of structures with similar overall shape/size but distinct features, thereby illustrating the kinetic trapping of multiple native-like solution structures in the gas phase.
AutoCCS: automated collision cross-section calculation software for ion mobility spectrometry-mass spectrometry
(Oxford University Press, 2021) Lee, Joon-Yong; Bilbao, Aivett; Conant, Christopher R.; Bloodsworth, Kent J.; Orton, Daniel J.; Zhou, Mowei; Wilson, Jesse W.; Zheng, Xueyun; Webb, Ian K.; Li, Ailin; Hixson, Kim K.; Fjeldsted, John C.; Ibrahim, Yehia M.; Payne, Samuel H.; Jansson, Christer; Smith, Richard D.; Metz, Thomas O.; Chemistry and Chemical Biology, School of Science
Motivation: Ion mobility spectrometry (IMS) separations are increasingly used in conjunction with mass spectrometry (MS) for separation and characterization of ionized molecular species. Information obtained from IMS measurements includes the ion's collision cross section (CCS), which reflects its size and structure and constitutes a descriptor for distinguishing similar species in mixtures that cannot be separated using conventional approaches. Incorporating CCS into MS-based workflows can improve the specificity and confidence of molecular identification. At present, there is no automated, open-source pipeline for determining CCS of analyte ions in both targeted and untargeted fashion, and intensive user-assisted processing with vendor software and manual evaluation is often required. Results: We present AutoCCS, an open-source software to rapidly determine CCS values from IMS-MS measurements. We conducted various IMS experiments in different formats to demonstrate the flexibility of AutoCCS for automated CCS calculation: (i) stepped-field methods for drift tube-based IMS (DTIMS), (ii) single-field methods for DTIMS (supporting two calibration methods: a standard and a new enhanced method) and (iii) linear calibration for Bruker timsTOF and non-linear calibration methods for traveling wave based-IMS in Waters Synapt and Structures for Lossless Ion Manipulations. We demonstrated that AutoCCS offers an accurate and reproducible determination of CCS for both standard and unknown analyte ions in various IMS-MS platforms, IMS-field methods, ionization modes and collision gases, without requiring manual processing. Availability and implementation: https://github.com/PNNL-Comp-Mass-Spec/AutoCCS. Supplementary information: Supplementary data are available at Bioinformatics online. Demo datasets are publicly available at MassIVE (Dataset ID: MSV000085979).
Experimental Determination of Activation Energies for Covalent Bond Formation via Ion/Ion Reactions and Competing Processes
(ACS, 2021) Cheung See Kit, Melanie; Shepherd, Samantha O.; Prell, James S.; Webb, Ian K.; Chemistry and Chemical Biology, School of Science
The combination of ion/ion chemistry with commercially available ion mobility/mass spectrometry systems has allowed rich structural information to be obtained for gaseous protein ions. Recently, the simple modification of such an instrument with an electrospray reagent source has allowed three-dimensional gas-phase interrogation of protein structures through covalent and noncovalent interactions coupled with collision cross section measurements. However, the energetics of these processes have not yet been studied quantitatively. In this work, previously developed Monte Carlo simulations of ion temperatures inside traveling wave ion guides are used to characterize the energetics of the transition state of activated ubiquitin cation/sulfo-benzoyl-HOAt reagent anion long-lived complexes formed via ion/ion reactions. The ΔH‡ and ΔS‡ of major processes observed from collisional activation of long-lived gas-phase ion/ion complexes, namely collision induced unfolding (CIU), covalent bond formation, or neutral loss of the anionic reagent via intramolecular proton transfer, were determined. Covalent bond formation via ion/ion complexes was found to be significantly lower energy compared to unfolding and bond cleavage. The ΔG‡ values of activation of all three processes lie between 55 and 75 kJ/mol, easily accessible with moderate collisional activation. Bond formation is favored over reagent loss at lower activation energies, whereas reagent loss becomes competitive at higher collision energies. Though the ΔG‡ values between CIU of a precursor ion and covalent bond formation of its ion/ion product complex are comparable, our data suggest covalent bond formation does not require extensive isomerization.
Experimental Evidence for Phosphorylation-Driven Allosteric Regulation of Alpha Synuclein Function
(bioRxiv, 2025-02-26) Dollar, Ashlyn N.; Webb, Ian K.; Chemistry and Chemical Biology, School of Science
Phosphorylation of serine 129 (pS129) in the intrinsically disordered protein alpha synuclein has long been associated with neurodegenerative disease. In the past several years, the functional relevance of pS219 has been uncovered by electrophysiology, immunoprecipitation, and proteomics as intricately connected with neurotransmitter release and synaptic vesicle (SV) cycling. Unexpectedly, binding to SNARE complex proteins VAMP-2 and synapsin only occurs with phosphorylation-competent alpha synuclein. The VAMP-2 binding domain has been shown to be residues 96-110, which does not include the phosphorylated residue, hinting at allosteric regulation of alpha synuclein protein-protein interactions by pS129. Within this study, cross-linking, covalent labeling, and collision induced unfolding of alpha synuclein and pS129 - as well as an additional encountered form in the brain, oxidized-M1, M5, M116, M127 alpha synuclein - are studied utilizing tandem mass spectrometry. Collision induced unfolding of proteins gives a fingerprint of the structures' relative compactness and stabilities of various conformations. Covalent labeling of proteins identifies solvent accessible residues and reveals the hydrophobicity (or hydrophilicity) of their microenvironment, while cross-linking of proteins maps the proximity of residue pairs. The combination of collision induced unfolding, covalent labeling, and cross-linking show unequivocally that phosphorylated-S129 alpha synuclein results in a more stable, more compact form. Our results provide evidence of an extensively folded amphipathic region that interacts strongly with the VAMP-2 binding domain. The phosphorylation-induced folding of the amphipathic region likely tunes other protein-protein interactions and interactions with SVs and membranes.
Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking
(Elsevier, 2021-05) Kit, Melanie Cheung See; Carvalho, Veronica V.; Vilseck, Jonah Z.; Webb, Ian K.; Chemistry and Chemical Biology, School of Science
Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure.
Multiplexed Conformationally Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation
(ACS, 2022-06) Chaturvedi, Ritu; Webb, Ian K.; Chemistry and Chemical Biology, School of Science
In this article, we present an approach for conformationally multiplexed, localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-IM-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. Gas-phase HDX with ECD fragmentation for exchange site localization was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed many regions with protection from HDX, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed high levels of deuterium incorporation throughout most of the protein sequence. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with unstructured regions of the protein relative to interior, structured regions, especially those involved in heme binding. With careful tuning and attention to deuterium scrambling, our approach holds promise for determining region-specific information on a conformer-selected basis for gas-phase protein structures, including localized characterizations of ligand, epitope, and protein–protein binding.
Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: Cytochrome C from neutral and acidic solutions
(Springer, 2023-02) Cain, Rebecca L.; Webb, Ian K.; Chemistry and Chemical Biology, School of Science
Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under “gentle” (i.e., low energy) ion transmission conditions with structures measured under “harsh” (i.e., higher energy) transmission. This work confirms that when using “gentle” instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, “harsh” conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.
Reactivities Leading to Potential Chemical Repair of Sunlight-Induced DNA Damage: Mechanistic Studies of Cyclobutane Pyrimidine Dimer (CPD) Lesions under Alkaline Conditions
(2020-12) Chaturvedi, Ritu; Long, Eric C.; McLeish, Michael J.; Pu, Jingzhi; Webb, Ian K.
Cyclobutane pyrimidine dimers (CPD) are the predominant DNA lesions formed upon exposure of this biopolymer to sunlight. Given the potentially dire biological consequences of DNA lesions, there is a need to fully characterize their behavior, with an eye towards understanding their complete reactivity and as a possible means to detect and quantify their presence in the genome. The work described in this dissertation describes studies of the alkaline reactivity of CPD lesions generated within dinucleotide & polynucleotide strands. It was found that CPD-TpT is generally inert under alkaline conditions at room temperature, which is in agreement with earlier studies on alkaline hydrolysis of CPD-thymine and CPD-thymidine. However, a re-evaluation of the same reaction in the presence of 18O labelled water demonstrated that, similar to other UV-induced DNA lesions containing a saturated pyrimidine ring, CPD undergoes a water addition at the C4=O group of the nucleobase leading to the formation of a hemiaminal intermediate. This intermediate, however, does not lead to hydrolysis products and completely reverts to starting material under those same conditions. Moreover, the two C4=O groups present on 3′ and 5′-thymines in a CPD molecule show different chemical reactivities, with the 3′ C4=O group having greater affinity towards water addition as compared to the one on 5′ end, a fact reflected in different rates of exchange with the incoming nucleophile leading to the hemiaminal intermediate. The 18O labelling reaction was also investigated in CPD lesions generated within oligonucleotides to probe the cause of asymmetry between the 3′ vs 5′ C4=O groups; ultimately, it was determined that the asymmetric reactivity observed to occur between the two C4=O groups was an intrinsic property of the CPD molecule and did not arise as a result of asymmetry in a dinucleotide setting. In addition to the above studies, during the course of the investigation of the nucleophilic reactivity of CPD, a chemical reaction was observed leading to what appeared to be the rapid and total chemical reversal of CPD lesions to the original TpT (thymine-thymine dinucleotide)! This “repair” reaction occurred when CPD reacted with hydrazine, and appears facilitated by an inert atmosphere under which it rapidly proceeds to completion at room temperature.
Recent Technological Developments for Native Mass Spectrometry
(Elsevier, 2022-01) Webb, Ian K.; Chemistry and Chemical Biology, School of Science
Native mass spectrometry (MS), the analysis of proteins and protein complexes from solutions that stabilize native solution structures, is a rapidly expanding area. There is strong evidence supporting the retention of proteins' native folds in the absence of solvent under the experimental timescales of MS experiments. Therefore, instrumentation has been developed to use gas-phase native-like protein ions to exploit the speed, sensitivity, and selectivity of mass spectrometry approaches to solve emerging problems in structural biology. This article reviews some of the recent advances and applications in gas-phase instrumentation for structural proteomics.

Browsing by Author "Webb, Ian K."

Results Per Page

Sort Options