Indiana University-Purdue University Indianapolis (IUPUI) / Gas-phase ion/ion covalent modification was coupled to ion mobility/mass spectrometry analysis to directly correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross-section (CCS) distributions were measured prior to ion/ion reactions to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Ubiquitin ions were electrosprayed from aqueous and methanolic solutions yielding a range of different charge states that were analyzed by ion mobility and time-of-flight mass spectrometry. Aqueous solutions stabilizing the native state of ubiquitin generated folded ubiquitin structures with CCS values consistent with the native state. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision-induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous solutions were covalently modified preferentially at the lysine 29 and arginine 54 residues, indicating that elements of secondary structure, as well as tertiary structure, were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol and low pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase.

The utility of 193 nm ultraviolet photodissociation (UVPD) and negative electron transfer dissociation (NETD) for the characterization of peptide anions was systematically evaluated. UVPD outperformed NETD in nearly all metrics; however, both methods provided complementary information to traditional collision induced dissociation (CID) of peptide cations in high throughput analyses. In order to enhance the performance of NETD, activated ion negative electron transfer dissociation (AI-NETD) methods were developed and characterized. The use of low-level infrared photoactivation or collisional activation during the NETD reaction period significantly improved peptide anion sequencing capabilities compared to NETD alone. Tyrosine deprotonation was shown to yield preferential electron detachment upon NETD or UVPD, resulting in N - C[alpha] bond cleavage N-terminal to the tyrosine residue. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages were regularly observed under high pH conditions. Transmission mode desorption electrospray ionization (TM-DESI) was coupled with 193 nm UVPD and CID for the rapid analysis and identification of protein digests. Comparative results are presented for TM-DESI-MS/CID and TM-DESI-MS/UVPD analyses of five proteolyzed model proteins. In some cases TM-DESI/UVPD outperformed TM-DESI-MS/CID due to the production of an extensive array of sequence ions and the ability to detect low m/z product ions. 193 nm UVPD was implemented in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa was achieved. The high-energy activation afforded by UVPD exhibited far less precursor ion charge state dependence than conventional methods, and the viability of 193 nm UVPD for high throughput top-down proteomics analyses was demonstrated for the less 30 kDa protein from a fractionated yeast cell lysate. The use of helium instead of nitrogen as the C-trap and HCD cell bath gas and trapping ions in the HCD cell prior to high resolution mass analysis significantly reduced the signal decay rate for large protein ions. As a result, monoclonal IgG1 antibody was isotopically resolved and mass accurately determined. A new high mass record for which accurate mass and isotopic resolution has been achieved (148,706.3391 Da ± 3.1 ppm) was established. / text

The advent of electrospray ionization enabled the study of intact protein complexes via MS. For example, in the mid-1990s, the observation that viruses can survive after entering the gas-phase and still retain activity was shown. Advances in sample preparation methodologies, mainly native MS, allowed for the preservation of large non-covalently bound complexes, which led to structural characterization studies that were previously unachievable. However, native MS suffers from complications arising from inherent heterogeneity and severe salt adduction. Consequently, the spectra can consist of broad and overlapping peaks that may even preclude the ability to obtain a mass measurement. This dissertation will focus on a gas-phase technique to address highly complex native MS scenarios that give rise to poorly resolved signals using the E. coli ribosome as one model system. Moreover, brief discussion of improvements made on our QToF platform (SCIEX 5600) will be compared with other state-of-the-art instruments. Lastly, other applications to our ion/ion reaction workflow will be explored.

Popular catalytic cycles, such as the Heck, Suzuki, and Negishi, utilize metal centers that oscillate between two oxidation states (II/0) during the three main steps of catalysis: reductive elimination, oxidative addition, and transmetallation. There has been a push to use less toxic, cheaper metal centers in catalytic cycles, leading to interest in first-row transition metals, such as nickel and cobalt. With these metals, the cycles can potentially pass through the +1 oxidation state, which acts as reactive intermediates, undergoing oxidative additions to form products, potentially with radical characteristics. The oxidative addition steps of catalytic cycles are critical to determining overall rates and products, however in many cases, these steps have not been amenable to study, in either condensed phase or gas phase, in the past. Through the use of electron transfer dissociation (ETD) technology on a modified Thermo Electron LTQ XLTM mass spectrometer, it is possible to generate intermediates in these catalytic cycles, including those in unusual oxidation states. Using sequentially coupled ion/ion-ion/molecule reactions, the reduced, reactive intermediate can be readily generated, isolated, and studied.As a model set of reactions, the mono- and bis-phenanthroline complexes of Fe(I), Co(I), Ni(I), Cu(I), and Zn(I) were formed by reduction of the corresponding M(II) species in an ion/ion reaction with the fluoranthenyl radical anion. The chemistry of the M(I) species was probed in ion/molecule reactions with allyl iodide. In order to explore ligand effects and the scope of oxidative addition reagents further, bipyridine and terpyridine were studied with these five first-row transition metal complexes while using an acetate series and other substrates for oxidative additions. Through these studies, the roles of the metal and ligand in dictating the product distributions and reaction rates were assessed. Metal electron count, ligand flexibility, and coordination number are critical factors. The overall reactivity is in accord with density functional theory calculations and mirrors that of proposed intermediates in condensed-phase catalytic cycles. In addition, second- and third-row transition metals (Ru(I), Pd(I), and Pt(I)) were explored with bipyridine, mono- and bis-triphenylphosphine, and 1,2-bis(diphenylphosphino)benzene ligation schemes. A variety of oxidative addition reagents were surveyed to determine the scope of reactivity and preference toward metal-carbon bond formation or carbon radical formation.

<p>Lipids are important cellular biomolecules that perform essential functional and biological roles. For instance, lipids in the cell are the compartmentalizer for the cytoplasm and an energy storage unit. The knowledge surrounding lipids is abundant, yet there is still so much to uncover. There are many categories of lipids and within each category the structural composition is extremely diverse. In turn, the dramatic structural complexity of lipids demands analytical methods capable of providing in-depth structural characterization of individual molecular structures. However, lipid structural elucidation has remained challenging, namely due to the presence of isomeric and isobaric species with a complex mixture. In particular, isomeric/isobaric lipid structures arise from variations in class, headgroup, fatty acyl chain, <i>sn</i>-position, and/or carbon-carbon double bond (C=C) position(s). Recently, recent research suggests C=C composition impacts lipid physical properties, metabolic fate, and intermolecular interactions. Thus, analytical strategies capable of localizing sites of unsaturation are of interest in the lipidomics community.</p> <p>Mass spectrometry (MS) is a leading tool for lipid analysis. Electrospray ionization (ESI), a soft ionization method, is the most commonly used method for lipid ionization as a means of taking the ions from liquid-phase to gas-phase without extensive decomposition of the species. Utilizing ESI-MS, lipids can be identified at a sum compositional level via accurate mass measurements. . With tandem mass spectrometers, lipid ions can be further probed, utilizing tandem-MS (MS/MS) to generate structurally informative product ion spectra that facilitate the assignment of lipid molecular structure. More so, gas-phase ion/ion reactions represent a unique MS-based technique that has improved the analysis of lipids structures. Gas-phase ion/ion reactions allow for lipid species to be charge inverted from one polarity to the opposite polarity. This reaction enables lipids to be ionized in a polarity that is optimal for class identification and further investigated in the opposite polarity where more structural information is obtained. All the information provided is captured without the requirement of multiple solution conditions which is necessary when analyzing in both polarities. In the case of charge inverted lipids from positive ion mode to negative ion mode, fatty acyl composition can be obtained; however, C=C information is lacking.</p> <p>MS can also be paired with other analytically technologies to assist with lipid analysis. One of those technologies is liquid chromatography (LC), which allows for the separation of lipids based on different characteristic depending on the column type being used. Reverse-phase LC (RPLC) allows for the separation of lipid molecular species based on structural composition. RPLC-MS/MS benefits from the ability to separate lipids and determine their fatty acyl chain composition but it is difficult to specify C=C location with the use of a synthetic standard that is identical to each molecular species being analyzed.</p> <p>Commonality between the gas-phase ion/ion reactions for charge inversion of lipids and RPLC-MS/MS approaches is the inability to provide C=C coverage. In-solution and unique ion activation techniques have been developed for seeking such information. The Paternò–Büchi reaction is a UV-initiated [2 + 2]-cycloaddition of an excited carbonyl containing compound onto an olefin group. This reaction can be initiated onto the alkene group within an unsaturated lipid aliphatic chain to form an oxetane ring modification. There are two product ions that can be formed upon each unsaturation site due to a lack of regioselectivity the reagent can attach at either side of the C=C. The modified lipids can be taken into gas-phase and collisionally activated via low-energy collision induced dissociation, generating product ions indictive of C=C position(s). The work herein shows the incorporation of the PB reaction into the gas-phases ion/ion reaction and RPLC-MS/MS apparatuses for C=C localization. The methods have been applied to the lipid extracts of bovine liver and human plasma for confident molecule species determination.</p>

<p> Mass spectrometry (MS) is one of the most commonly used analytical techniques in bioanalytical analysis, allowing scientists to characterize molecules with very diverse chemical features. The advance in ionization strategies significantly improves the potential in using MS for that purpose, especially electrospray ionization (ESI) can generate ions directly from solution in ambient conditions, showing high flexibility in coupling with other techniques. Moreover, a hallmark of the ESI of large polymeric molecules is also its tendency to generate a distribution of charge states based on their chemical characteristics, allowing us to exploit the multiple charging phenomenon in various applications. </p> <p>This dissertation introduces the relationships between ESI and multiple charging phenomena with different proposed ionization models, and how condensed-phase and gas-phase approaches affect the multiple charging phenomenon. Moreover, multiply charged ions permit gas-phase ion/ion reactions to occur without neutralizing the ions. Therefore, various ion/ion reactions can be utilized for distinct analytical purposes. Objectively, this dissertation focuses on the investigation of the multiple charging phenomenon from ESI-MS, and the applications from taking the multiply charged ions to perform gas-phase ion chemistry in order to a) manipulate the charges of the targeted ions; b) invert the polarity of the targeted ions; c) and characterization of the ions from the gas-phase ion/ion reactions.</p> <p> The first work demonstrates how multiple components (i.e., complicated mixtures) lead to a highly congested spectrum of ions with overlapped m/z values, resulting from the multiple charging phenomenon after the ESI process. Utilizing ionic reactions can de-congest the spectra via manipulating the charges of the ions to separate the overlapped signals. A universal spectral pattern in the ESI mass spectra is observed while analyzing multiply-charged homopolymers. Various parameters, such as the charges of the ions, widths of polymer distributions, monomer mass, and cationizing agent masses, are investigated to show how they can affect the appearance of the unique patterns, which condense the information of the overall distribution of the homopolymers. Combined with gas-phase charge reduction (i.e., proton transfer reaction), we can characterize the size distribution of polydisperse homopolymer samples.</p> <p>Second, a novel type charge inversion ion/ion reaction summarizing the conversion of multiply charged protein ions to their opposite polarity and still holds multiple charges is reported. The reaction occurs via a single ion/ion collision with highly charged reagent ions, which we usually obtain from biological relevant polymers. Hyaluronic acid (HAs) anions and polyethylenimine (PEI) cations are used as the charge inversion reagents to react with protein ions. Remarkably, inversion of high absolute charge (up to 41) from the reaction is demonstrated. All mechanisms for ion/ion charge inversion involve low-energy ions proceeding via the formation of a long-lived complex. Factors that underlie the charge inversion of protein ions to the opposite polarity with high charge states in reaction with those reagent ions are hypothesized to include: (i) the relatively high charge densities of the HA anions and PEI cations that facilitate the extraction/donation of multiple protons from/to the protein leading to multiply charged protein anions/cations, (ii) the relatively high sum of absolute charges of the reactants that leads to high initial energies in the ion/ion complex, and (iii) the relatively high charge of the ion/ion complex following the multiple proton transfers that tends to destabilize the complex.</p> <p>Third, shotgun MS strategies coupled with different gas-phase ion chemistry and tandem MS to analyze glycolipids are demonstrated. Glycolipids contain both carbohydrates and lipids structure components that it is incredibly challenging to analyze with MS. Isomeric cerebrosides (n-HexCer) and glycosphingosines (n-HexSph), which hold isomerisms in diastereomeric sugar head groups (glucose and galactose), anomeric glycosidic linkages (alpha- or beta-), and isomeric amide-bonded monounsaturated fatty acyl chain (double bond location) are successfully differentiated by dissociating gas-phase ion/ion reaction products, the charge-inverted complex cations. Both relative and absolute quantification of the isomers is also achieved, and analytical performances are evaluated in terms of accuracy, precision, and inter-day precision, allowing us to perform mixture analysis. Porcine brains were used to demonstrate the ability to profile and quantify those isomers from biological extracts. Moreover, a parallel workflow is also proposed for gangliosides, which have more complicated structures among their glycan moiety. Metal cation transfer, proton transfer, and charge inversion reactions are utilized to manipulate the ion types to provide better structural information. The proposed workflow allows us to clean up the mass spectra by neutralizing interfering isobaric ions, differentiate isomeric gangliosides, and perform relative quantitation when the standards are available. The workflow also is used to obtain gangliosides profiles from biological matrices. Overall, work in this dissertation takes advantage of the multiple charging phenomenon and couples with gas-phase ion/ion reactions to achieve various analyses among a wide range of biological-related samples.</p>

<p dir="ltr">Originating from J. J. Thomsons original work and the development of electrospray ionization (ESI) by John B. Fenn, mass spectrometry offers a versatile analytical tool to measure beyond an ion’s m/z, especially for biomolecules. Gas-phase ion/ion reactions within a mass spectrometer offers an attractive approach to study biomolecules as they take place on the millisecond and sub millisecond time scale, have high efficiency, allow oppositely charged ions to interact with each other in a controlled manner, and a allows for selection of each reactant prior to the reaction via ion isolation. This can be used to probe gas-phase chemistry that can reflect reactions in solution, however gas-phase reactions have no solvent effects and happen faster, making it a simpler experiment. Here, a variety of gas-phase ion/ion reactions and ion trap methodologies are described to study mostly lipids with a minor amount of transmetalation at the beginning.</p><p dir="ltr">First, a series of multivalent metals complexed to neutral ligands are demonstrated to form ion-pairs with tetraphenylborate anions via ion/ion reactions. The resulting products were subjected to collision induced activation (CID) to observe their involvement in transmetalation, complementary density functional theory (DFT) calculations are provided as well. Next, sequential ion/ion reactions were performed to convert isomeric phosphoinositol phosphates dianions to monocations to reveal structural characterization and isomeric differentiation utilizing tandem MS and dissociation kinetics. The following two chapters after, reports on complementary efforts to separate lipids in the gas-phase of different mass and charge but similar mass-to-charge (m/z) resulting in overlapping m/z signals. The first report demonstrates a physical approach where singly and double charged lipids are separated in space from each other, trapped simultaneously such that no information is lost. The second utilizes a lanthanide, Yb3+ trication complex that underwent ion/ion reactions with singly and doubly charged lipid anions of similar m/z that result in different m/z products for each singly and doubly charged lipids. Lastly, a sequential ion/ion approach utilizing hexa(ethylene glycol) dithiol as a novel reagent to charge invert structurally uninformative lipid cations to structurally informative anions with subsequent carbon-carbon double bond localization.</p>