Studies of the Fragmentation Mechanisms of Deprotonated Lignin Model Compounds in Tandem Mass Spectrometry.

Unlabeled and deuterium-labeled dimeric lignin model compounds with ß-O-4 linkages were evaporated and ionized using negative ion mode electrospray ionization, transferred into a linear quadrupole ion trap, isolated, and subjected to collision-activated dissociation (CAD; MS2 experiments). The elemental compositions of the fragment ions were determined by using a high-resolution Orbitrap mass analyzer, and their structures were examined using further CAD experiments (MSn experiments wherein n = 2-5). Data analysis was facilitated by determining the fragmentation pathways for several deprotonated model compounds. The structures of the key fragment ions of several pathways were determined by comparison of the CAD mass spectra measured for undeuterated and deuterated analogues and for deprotonated authentic compounds. Some of the proposed reaction mechanisms were tested by examining additional deprotonated synthetic model compounds. Quantum chemical calculations were used to delineate the most likely reaction pathways and reaction mechanisms. This work provides basic information needed for the design of tandem mass spectrometry-based CAD sequencing strategies for mixtures of lignin degradation products.

Factors Affecting the Limit of Detection for HPLC/Tandem Mass Spectrometry Experiments Based on Gas-Phase Ion-Molecule Reactions.

Diagnostic and predictable gas-phase ion-molecule reactions have emerged as a potential alternative to collision-activated dissociation in tandem mass spectrometry (MS2) experiments performed to gain structural information for unknown organic compounds, such as drug metabolites, in complex mixtures. However, the applicability of this approach for analyzing metabolites at physiologically relevant concentrations has not been determined. In this study, HPLC/MS2 experiments based on gas-phase ion-molecule reactions of protonated model compounds were successfully conducted at nanomolar and picomolar analyte concentrations. As the analyte concentration decreased, the signal-to-noise ratio of the HPLC peaks decreased more than the signal-to-noise ratio of the mass spectrometer peaks. Therefore, the HPLC part of this analysis was the primary limiting factor for each analyte (rather than the ion-molecule reactions). The ion-molecule reaction limits of detection ranged from 50 pM to 250 nM with the average being 50-100 nM. Since all compounds had ion-molecule reaction detection limits below 500 nM, the detection limits are within the physiologically relevant range for in vivo studies of drugs and drug metabolites. When considering only mass spectrometry, the number of ion isolation events (one in MS2 experiments involving ion-molecule reactions or two in MS3 experiments involving CAD of products formed upon ion-molecule reactions) and the subsequent CAD in the MS3 experiments were the most important limiting factors. Indeed, the limit of detection for the MS3 experiments was 250 nM, about three times higher than the average ion-molecule reaction detection limit of 75 nM but still within physiologically relevant concentrations.

Analyzing and Tuning the Chalcogen-Amine-Thiol Complexes for Tailoring of Chalcogenide Syntheses.

The amine-thiol solvent system has been used extensively to synthesize metal chalcogenide thin films and nanoparticles because of its ability to dissolve various metal and chalcogen precursors. While previous studies of this solvent system have focused on understanding the dissolution of metal precursors, here we provide an in-depth investigation of the dissolution of chalcogens, specifically Se and Te. Analytical techniques, including Raman, X-ray absorption, and NMR spectroscopy and high-resolution tandem mass spectrometry, were used to identify pathways for Se and Te dissolution in butylamine-ethanethiol and ethylenediamine-ethanethiol solutions. Se in monoamine-monothiol solutions was found to form ionic polyselenides free of thiol ligands, while in diamine-monothiol solutions, thiol coordination with polyselenides was predominately observed. When the relative concentration of thiol is increased to that of Se, the chain length of polyselenide species was observed to shorten. Analysis of Te dissolution in diamine-thiol solutions also suggested the formation of relatively unstable thiol-coordinated Te ions. This instability of Te ions was found to be reduced by codissolving Te with Se in diamine-thiol solutions. Analysis of the codissolved solutions revealed the presence of atomic interaction between Se and Te through the identification of Se-Te bonds. This new understanding then provided a new route to dissolve otherwise insoluble Te in butylamine-ethanethiol solutions by taking advantage of the Se2- nucleophile. Finally, the knowledge gained for chalcogen dissolutions in this solvent system allowed for controlled alloying of Se and Te in PbSenTe1-n material and also provided a general knob to alter various metal chalcogenide material syntheses.

Differentiation of Deprotonated Acyl-, N-, and O-Glucuronide Drug Metabolites by Using Tandem Mass Spectrometry Based on Gas-Phase Ion-Molecule Reactions Followed by Collision-Activated Dissociation.

Glucuronidation, a common phase II biotransformation reaction, is one of the major in vitro and in vivo metabolism pathways of xenobiotics. In this process, glucuronic acid is conjugated to a drug or a drug metabolite via a carboxylic acid, a hydroxy, or an amino group to form acyl-, O-, and/or N-glucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway. However, some acyl-glucuronides react with biomolecules in vivo, which may result in immune-mediated idiosyncratic drug toxicity (IDT). In order to avoid this, one may attempt in early drug discovery to modify the lead compounds in such a manner that they then have a lower probability of forming reactive acyl-glucuronide metabolites. Because most drugs or drug candidates bear multiple functionalities, e.g., hydroxy, amino, and carboxylic acid groups, glucuronidation can occur at any of those. However, differentiation of isomeric acyl-, N-, and O-glucuronide derivatives of drugs is challenging. In this study, gas-phase ion-molecule reactions between deprotonated glucuronide metabolites and BF3 followed by collision-activated dissociation (CAD) in a linear quadrupole ion trap mass spectrometer were demonstrated to enable the differentiation of acyl-, N-, and O-glucuronides. Only deprotonated N-glucuronides and deprotonated, migrated acyl-glucuronides form the two diagnostic product ions: a BF3 adduct that has lost two HF molecules, [M - H + BF3 - 2HF]-, and an adduct formed with two BF3 molecules that has lost three HF molecules, [M - H + 2BF3 - 3HF]-. These product ions were not observed for deprotonated O-glucuronides and unmigrated, deprotonated acyl-glucuronides. Upon CAD of the [M - H + 2BF3 - 3HF]- product ion, a diagnostic fragment ion is formed via the loss of 2-fluoro-1,3,2-dioxaborale (MW of 88 Da) only in the case of deprotonated, migrated acyl-glucuronides. Therefore, this method can be used to unambiguously differentiate acyl-, N-, and O-glucuronides. Further, coupling this methodology with HPLC enables the differentiation of unmigrated 1-ß-acyl-glucuronides from the isomeric acyl-glucuronides formed upon acyl migration. Quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory were employed to probe the mechanisms of the reactions of interest.

Click chemistry assisted single-molecule fingerprinting reveals a 3D biomolecular folding funnel.

A 3D folding funnel was proposed in the 1990s to explain the fast kinetics exhibited by a biomacromolecule in presence of seemingly unlimited folding pathways. Over the years, numerous simulations have been performed with this concept; however, experimental verification is yet to be attained even for the simplest proteins. Here, we have used a click chemistry based strategy to introduce six pairs of handles in a human telomeric DNA sequence. A laser-tweezers-based, single-molecule structural fingerprinting on the six inter-handle distances reveals the formation of a hybrid-1 G-quadruplex in the sequence. Kinetic and thermodynamic fingerprinting on the six trajectories defined by each handle-pair depict a 3D folding funnel and a kinetic topology in which the kinetics pertaining to each handle residue is annotated for this G-quadruplex. We anticipate the methods and the concepts developed here are well applicable to other biomacromolecules, including RNA and proteins.