Collision-induced Dissociation Research Articles

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer.

The onset and progression of cancer is associated with changes in the composition of the lipidome. Therefore, better understanding of the molecular mechanisms of these disease states requires detailed structural characterization of the individual lipids within the complex cellular milieu. Recently, changes in the unsaturation profile of membrane lipids have been observed in cancer cells and tissues, but assigning the position(s) of carbon-carbon double bonds in fatty acyl chains carried by membrane phospholipids, including the resolution of lipid regioisomers, has proven analytically challenging. Conventional tandem mass spectrometry approaches based on collision-induced dissociation of ionized glycerophospholipids do not yield spectra that are indicative of the location(s) of carbon-carbon double bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have emerged as alternative ion activation modalities wherein diagnostic product ions can enable de novo assignment of position(s) of unsaturation based on predictable fragmentation behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra are acquired on the same mass spectrometer to evaluate the relative performance of the two modalities for lipid identification and to interrogate the respective fragmentation pathways under comparable conditions. Based on investigations of lipid standards, fragmentation rules for each technique are expanded to increase confidence in structural assignments and exclude potential false positives. Parallel application of both methods to unsaturated phosphatidylcholines extracted from isogenic colorectal cancer cell lines provides high confidence in the assignment of multiple double bond isomers in these samples and cross-validates relative changes in isomer abundance.

A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines.

Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.

Tandem Mass Spectrometry Approaches for Differentiation and Quantification Pidotimod and Its Three Isomers in the Gas Phase.

Pidotimod is a chiral drug that possesses two chiral centers, resulting in three isomeric impurities (analytes, A). This study employs electrospray ionization ion trap mass spectrometry (ESI-MS) through collision-induced dissociation (CID) to investigate the chiral recognition of pidotimod and its three isomers to eliminate chromatographic separation. Three approaches were explored: (1) Protonated molecules in CID exhibited discriminative potential for diastereomers, with the ability to distinguish between S,S and R,R configurations, albeit with an Rchiral value of ~1.8. However, differentiation between R,S and S,R configurations was not achievable. (2) Alkali adductions (lithium and sodium) only discerned diastereomers. The Rchiral values of the diastereomers obtained from alkali adduct ions were significantly lower than those obtained from protonated ions. (3) Therefore, a third approach was used to address the challenge of distinguishing between R,S and S,R configurations, including the introduction of chiral references (ref) and transition metals (MII) to form metal-bound complexes [MII(A)(ref)-H]+. Additionally, we synthesized a novel ligand, 4-(N-tert-butoxycarbonyl [Boc]-L-prolinamido)phenol (denoted as ligand A), by modifying N-t-Boc-L-Pro with 2-aminophenol, which, in combination with CuII and NiII, enabled simultaneous differentiation of all four isomers. CuII complexes exhibited significant chiral selectivity between R,S and S,R configurations. Density functional theory calculations were performed to further elucidate the stereodynamic behavior and stoichiometry of these ions in the gas phase. These calculations revealed the interaction energy and coordination sites of the precursor ions in the gas phase, correlating well with MS/MS experiment results. Additionally, the logarithm of the CuII complexes' characteristic fragment ion abundance ratio demonstrated a strong linear relationship with enantiomeric excess (ee). This study presents a novel strategy for chiral drug quality control that eliminates chromatographic separation.

Structural characterization and bioactivity profiling of the fungal peptaibiotic tolypin reveal protective effects against influenza viruses.

In a bioprospection for new antivirals, we tested nonribosomally biosynthesized polypeptide antibiotics in MDCK II cells for their actions on influenza A and B viruses (IAV/IBV). Only tolypin, a mixture of closely related 16-residue peptaibiotics from the fungus Tolypocladium inflatum IE 1897, showed promising activity. It was selected for further investigation and structural characterization byultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HR-MS/MS) and ultrahigh performance liquid chromatography coupled to in-source collision-induced dissociation tandem mass spectrometry (UHPLC-isCID-HR-MS/MS), revealing 12 partially co-eluting individual peptides that were fully sequenced. Since tolypin-related efrapeptins are potent inhibitors of F1/Fo-ATPase, we screened tolypin for its toxicity against MDCK II cells and larvae of the greater wax moth Galleria mellonella. We found that a nontoxic concentration of tolypin (1 µg/mL) reduced the titer of two IBV strains by 4-5 log values, and that of an H3N2 strain by 1-2 log values, but the H1N1pdm strain was not affected. The higher concentrations of tolypin were cytostatic to MDCK II cells, shifted their metabolism from oxidative phosphorylation to glycolysis, and induced paralysis in G. mellonella, supporting the inhibition of F1/Fo-ATPase as the mode of action. Our results lay the foundations for future work to investigate the interplay between viral replication and cellular energy metabolism, as well as the development of drugs that target host factors.

Energy-Resolved In-Source Collison-Induced Dissociation for Isomer Discrimination.

While mass spectrometry remains a gold-standard tool for analyte detection, characterization, and quantitation, isomer differentiation is often a challenge. Tandem mass spectrometry is a common approach to increase the selectivity of mass spectrometry and energy-resolved measurements can provide further improvements. However, not all mass spectrometers, especially those that are very compact and affordable, are amenable to such experiments. For instance, single-stage mass spectrometers with soft ionization provide no dissociation information and quadrupole ion trap instruments with resonant excitation do not necessarily provide as informative of energy-resolved curves, for instance when extensive sequential dissociation is responsible for much of the "fingerprint". In-source collision-induced dissociation (IS-CID) is one approach to overcoming these barriers to exploit the analytical selectivity of energy-resolved CID without the need for additional instrumentation; this approach could broaden the reach of these selectivity gains to additional user bases (e.g., educational settings, field portable devices). Here, we specifically investigate energy-resolved IS-CID with the goal of (1) comparing between energy-resolved appearance curves measured with true tandem mass spectrometry on a quadrupole time-of-flight instrument and those obtained using IS-CID, (2) evaluating the approach as a means of differentiating isomers/isobar sets, especially those with similar dissociation patterns, and (3) exploring additional analytical considerations relevant to method development and implementation. This proof-of-concept work establishes the analytical potential of this approach, opening doors for future method development for specific applications.

Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.

Cryogenic Photoelectron Spectroscopic and Theoretical Study of the Electronic and Geometric Structures of Undercoordinated Osmium Chloride Anions OsCln- (n = 3-5).

A series of anionic transition metal halides, OsCln- (n = 3-5), have been investigated using a newly developed, home-constructed, cryogenic anion cluster photoelectron spectroscopy. The target anionic species are generated through collision-induced dissociation in a two-stage ion funnel. The measured vertical detachment energies (VDEs) are 3.48, 4.54, and 4.81 eV for n = 3, 4, and 5, respectively. Density functional theory calculations at the B3LYP-D3(BJ)//aug-cc-pVTZ(-pp) level predict the lowest energy structures of the atomic form of OsCln- (n = 3-5) to be a quintet triangle, quartet square, and quintet square-based pyramid, respectively. The CCSD(T)-calculated VDEs and corresponding adiabatic detachment energies agree well with our experimental measurements. Analysis of the corresponding frontier molecular orbitals and charge density differences suggests that the d-orbitals of the transition metal Os play a primary role in the single-photon detachment processes, and the detached electrons originating from different molecular orbitals are distinguishable.

Modeling collisional kinetic energy damping, heating, and cooling of ions in mass spectrometers: A tutorial perspective

Many powerful methods in mass spectrometry rely on activation of ions by high-energy collisions with gas particles. For example, multiple Collision Induced Dissociation (CID) has been used for many years to determine structural information for ions ranging from small organics to large, native-like protein complexes. More recently, Collision Induced Unfolding (CIU) has proved to be a very powerful method for understanding high-order protein structure and detecting differences between similar proteins. Quantifying the thermochemistry underlying dissociation/unfolding in these experiments can be quite challenging without reliable models of ion heating and cooling. Established physical models of CID are valuable in predicting ion heating but do not explicitly include mechanisms for cooling, which may play a large part in CID/CIU in modern instruments. Ab initio and Molecular Dynamics methods are extremely computationally expensive for modeling CID/CIU of large analytes such as biomolecular ions. In this tutorial perspective, limiting behaviors of ion kinetic energy damping, heating, and cooling set by “extreme” cases are explored, and an Improved Impulsive Collision Theory and associated software (“Ion Simulations of the Physics of Activation”, IonSPA) are introduced that can model all of these for partially inelastic collisions. Finally, examples of modeled collisional activation of native-like protein ions under realistic experimental conditions are discussed, with an outlook toward the use of IonSPA in accessing the thermochemical information hidden in CID breakdown curves and CIU fingerprints.

DFT computational and mass spectrometry studies of intermolecular interaction of β-cyclodextrin with nifedipine

A systematic investigation of intermolecular interactions of β- cyclodextrin(β-CD)and Nifedipine(Nif)in gas phase and in water were done by the mass spectrometry and DFT calculation method. The results obtained clearly indicated that the aromatic ring containing the nitro moiety of Nifedipine inverted into the wider rim of β cyclodextrin, the high collision induced dissociation (CID) energy in mass spectrometry experiments demonstrated the stability of the 1:1 Nif-β-CD complexes. Compared to the native β-CD, HP- β-CD and DM- β-CD are more easily to form complexes with Nif due to their approximately double lower binding energy, the thermodynamic parameters, HOMO, LUMO, the global reactivity descriptors of these complexes were calculated and interpreted. Furthermore, the study of natural bonding orbitals (NBO) and quantum theory of protons in molecules (QTAIM) supports the existence of various hydrogen bonds and van der Waals interactions in three complex structures, as predicted by chemical bonding principles. IGMH analysis give the visualized origin of weak attractive interactions between the CDs and the Nifedipine.

Applications of native mass spectrometry and ultraviolet photodissociation in protein structure and interaction analysis

Dynamic changes in the structures and interactions of proteins are closely correlated with their biological functions. However, the precise detection and analysis of these molecules are challenging. Native mass spectrometry (nMS) introduces proteins or protein complexes into the gas phase by electrospray ionization, and then performs MS analysis under near-physiological conditions that preserve the folded state of proteins and their complexes in solution. nMS can provide information on stoichiometry, assembly, and dissociation constants by directly determining the relative molecular masses of protein complexes through high-resolution MS. It can also integrate various MS dissociation technologies, such as collision-induced dissociation (CID), surface-induced dissociation (SID), and ultraviolet photodissociation (UVPD), to analyze the conformational changes, binding interfaces, and active sites of protein complexes, thereby revealing the relationship between their interactions and biological functions. UVPD, especially 193 nm excimer laser UVPD, is a rapidly evolving MS dissociation method that can directly dissociate the covalent bonds of protein backbones with a single pulse. It can generate different types of fragment ions, while preserving noncovalent interactions such as hydrogen bonds within these ions, thereby enabling the MS analysis of protein structures with single-amino-acid-site resolution. This review outlines the applications and recent progress of nMS and UVPD in protein dynamic structure and interaction analyses. It covers the nMS techniques used to analyze protein-small-molecule ligand interactions, the structures of membrane proteins and their complexes, and protein-protein interactions. The discussion on UVPD includes the analysis of gas-phase protein structures and interactions, as well as alterations in protein dynamic structures, and interactions resulting from mutations and ligand binding. Finally, this review describes the future development prospects for protein analysis by nMS and new-generation advanced extreme UV light sources with higher brightness and shorter pulses.

Hydration energies of calcium hydroxide cation, CaOH+(H2O)x (x = 1–6), studied using guided ion beam tandem mass spectrometry

Hydration energies of CaOH+(H2O)x, x = 1–6, were obtained using threshold collision-induced dissociation (TCID) with xenon (Xe) as conducted with a guided ion beam tandem mass spectrometer (GIBMS). The primary reaction pathway observed for all complexes is the loss of one water ligand, followed by the loss of additional water molecules at higher collision energies for x > 1. The kinetic-energy-dependent cross sections for dissociation of CaOH+(H2O)x complexes were modeled to obtain 0 K binding energies after accounting for lifetime effects, energy distributions, and pressure effects. Except for x = 5, experimental threshold energies measured through TCID agree well with theoretical hydration energies determined using B3LYP/6-311+G(d,p) structure geometries followed by single point energies calculated at B3LYP, B3P86, M06, and MP2(full) levels of theory with a 6-311+G(2d,2p) basis set. B3LYP-GD3BJ and ωB97XD calculations were also used, with the former yielding results having the best agreement with the threshold energies extracted from the analysis of the TCID cross sections.

Quantitative proteome-wide O-glycoproteomics analysis with FragPipe.

Identification of O-glycopeptides from tandem mass spectrometry data is complicated by the near complete dissociation of O-glycans from the peptide during collisional activation and by the combinatorial explosion of possible glycoforms when glycans are retained intact in electron-based activation. The recent O-Pair search method provides an elegant solution to these problems, using a collisional activation scan to identify the peptide sequence and total glycan mass, and a follow-up electron-based activation scan to localize the glycosite(s) using a graph-based algorithm in a reduced search space. Our previous O-glycoproteomics methods with MSFragger-Glyco allowed for extremely fast and sensitive identification of O-glycopeptides from collisional activation data but had limited support for site localization of glycans and quantification of glycopeptides. Here, we report an improved pipeline for O-glycoproteomics analysis that provides proteome-wide, site-specific, quantitative results by incorporating the O-Pair method as a module within FragPipe. In addition to improved search speed and sensitivity, we add flexible options for oxonium ion-based filtering of glycans and support for a variety of MS acquisition methods and provide a comparison between all software tools currently capable of O-glycosite localization in proteome-wide searches.

Studying Cation Exchange in {Cr7Co} Pseudorotaxanes: Preparatory Studies for Making Hybrid Molecular Machines.

In the design of dynamic supramolecular systems used in molecular machines, it is important to understand the binding preferences between the macrocycle and stations along the thread. Here, we apply 1H NMR spectroscopy to investigate the relative stabilities of a series of linear alkylammonium templated pseudorotaxanes with the general formula [H2NRR'][Cr7CoF8(O2CCH2 tBu)16] by exchanging the cation in solution. Our results show that the pseudorotaxanes are able to exchange threads via a dissociative mechanism. The position of equilibrium is dependent upon the ammonium cation and solvent used. Short chain primary ammonium cations are shown to be far less favourable macrocycle stations than secondary ammonium cations. Collision-induced dissociation mass spectrometry (CID-MS) has been used to look at disassembly of the pseudorotaxanes in a solvent-free environment and stability trends compared to those in acetone-d6. The energy needed to induce 50 % of the precursor ion loss (E50) is used and shows a similar trend to the equilibria measured by NMR. The relative stabilities of these hybrid inorganic-organic pseudo-rotaxanes are different to those of host-guest compounds involving crown ethers and this may be valuable for the design of molecular machines.

Nucleoside Cation Radicals: Generation, Radical-Induced Hydrogen Atom Migrations, and Ribose Ring Cleavage in the Gas Phase.

Nucleoside ions that were furnished on ribose with a 2'-O-acetyl radical group were generated in the gas phase by multistep collision-induced dissociation of precursor ions tagged with radical initiator groups, and their chemistry was investigated in the gas phase. 2'-O-Acetyladenosine cation radicals were found to undergo hydrogen transfer to the acetoxyl radical from the ribose ring positions that were elucidated using specific deuterium labeling of 1'-H, 2'-H, and 4'-H and in the N-H and O-H exchangeable positions, favoring 4'-H transfer. Ion structures and transition-state energies were calculated by a combination of Born-Oppenheimer molecular dynamics and density functional theory and used to obtain unimolecular rate constants for competitive hydrogen transfer and loss of the acetoxyl radical. Migrations to the acetoxyl radical of ribose hydrogens 1'-H, 2'-H, 3'-H, and 4'-H were all exothermic, but product formation was kinetically controlled. Both Rice-Ramsperger-Kassel-Marcus (RRKM) and transition-state theory (TST) calculations indicated preferential migration of 4'-H in a qualitative agreement with the deuterium labeling results. The hydrogen migrations displayed substantial isotope effects that along with quantum tunneling affected the relative rate constants and reaction branching ratios. UV-vis action spectroscopy indicated that the cation radicals from 2'-O-acetyladenosine consisted of a mixture of isomers. Radical-driven dissociations were also observed for protonated guanosine, cytosine, and thymidine conjugates. However, for those nucleoside ions and cation radicals, the dissociations were dominated by the loss of the nucleobase or formation of protonated nucleobase ions.

Chemokine Oligomers and the Impact of Fondaparinux Binding.

Heparin, a widely used clinical anticoagulant, is generally well-tolerated; however, approximately 1% of patients develop heparin-induced thrombocytopenia (HIT), a serious side effect. While efforts to understand the role of chemokines in HIT development are ongoing, certain aspects remain less studied, such as the stabilization of chemokine oligomers by heparin. Here, we conducted a combined ion mobility-native mass spectrometry study to investigate the stability of chemokine oligomers and their complexes with fondaparinux, a synthetic heparin analog. Collision-induced dissociation and unfolding experiments provided clarity on the specificity and relevance of chemokine oligomers and their fondaparinux complexes with varying stoichiometries, as well as the stabilizing effects of fondaparinux binding.

Explore the feasibility of electrospray ionization high-resolution mass spectrometry in elemental analysis

As a representative soft ionization technique, electrospray ionization (ESI) is commonly applied for the analysis of organic and biological compounds and is less used in inorganic applications. This study introduces the performance of ESI high-resolution mass spectrometry (HRMS) in the detection of elements. The HRMS instrument was equipped with a sub-atmospheric pressure (SAP) ESI source to reduce sample consumption and simplify the sampling operations. In addition, in-source collision-induced dissociation (IS-CID) was introduced into the analysis to generate simple metallic ion species such as atomic ions and charged oxides and reduce organic peak interferences. Various solutions containing metal salts and organometallic compounds were analyzed, demonstrating a picogram-level absolute detection limit and accurate multi-element identification capability of the instrument. Furthermore, SAP-ESI-HRMS exhibits diverse analytical performances, enabling both elemental and organic analyses by simply regulating the IS-CID energy in the measurement.

Recent additions and access to a multidimensional lipidomic database containing liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry information.

The importance of lipids in biology continues to grow with their recent linkages to more diseases and conditions, microbiome fluctuations, and environmental exposures. These associations have motivated researchers to evaluate lipidomic changes in numerous matrices and studies. Lipidomic analyses, however, present numerous challenges as lipid species have broad chemistries that require different extraction methods and instrumental analyses to evaluate and separate their many isomers and isobars. Increasing knowledge about different lipid characteristics is therefore crucial for improving their separation and identification. Here, we present a multidimensional database for lipids analyzed on a platform combining reversed-phase liquid chromatography, drift tube ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (RPLC-DTIMS-CID-MS). This platform and the different separation characteristics it provides enables more confident lipid annotations when compared to traditional tandem mass spectrometry platforms, especially when analyzing highly isomeric molecules such as lipids. This database expands on our previous publication containing only human plasma and bronchoalveolar lavage fluid lipids and provides experimental RPLC retention times, IMS collision cross section (CCS) values, and m/z information for 877 unique lipids from additional biofluids and tissues. Specifically, the database contains 1504 precursor [M + H]+, [M + NH4]+, [M + Na]+, [M-H]-, [M-2H]2-, [M + HCOO]-, and [M + CH3COO]- ion species and their associated CID fragments which are commonly targeted in clinical and environmental studies, in addition to being present in the chloroform layer of Folch extractions. Furthermore, this multidimensional RPLC-DTIMS-CID-MS database spans 5 lipid categories (fatty acids, sterols, sphingolipids, glycerolipids, and glycerophospholipids) and 24 lipid classes. We have also created a webpage (tarheels.live/bakerlab/databases/) to enhance the accessibility of this resource which will be populated regularly with new lipids as we identify additional species and integrate novel standards.

Sequencing of Phosphopeptides Using a Sequential Charge Inversion Ion/Ion Reaction and Electron Capture Dissociation Workflow.

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization.

Toward Designing Reactive Metal Clusters for Dinitrogen Activation: A Guideline Based on N2 Initial Adsorption.

Gas-phase metal clusters are ideal models to explore transition-metal-mediated N2 activation mechanism. However, the effective design and search of reactive clusters in N2 activation are currently hindered by the lack of clear guidelines. Inspired by the Sabatier principle, we discovered in this work that N2 initial adsorption energy (ΔEads) is an important parameter to control the N2 activation reactivity of metal clusters in the gas phase. This mechanistic insight obtained from high-level calculations rationalizes the N2 activation reactivity of many previously reported metal clusters when combined with the known factor determining the N≡N cleavage process. Furthermore, based on this guideline of ΔEads, we successfully designed several new reactive clusters for cleaving N≡N triple bond under mild conditions, including FeV2S2-, TaV2C2-, and TaV2C3-, the high N2 activation reactivity of which has been fully corroborated in our gas phase experiments employing mass spectrometry with collision-induced dissociation. The importance of ΔEads revealed in this work not only reshapes our understanding of N2 activation reactions in the gas phase but also could have implication for other N2 activation processes in the condensed phase. The more general establishment of this new perspective on N2 activation reactivity warrants future experimental and computational studies.

Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.

Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 μL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.