<p>Mass spectrometry-based gas-phase ion/ion reactions have grown considerably in the last decade. Their applications range from structural elucidation, instrument calibration, and spectral deconvolution. One field that has been amenable to these methods is proteomic studies. Proteins and peptides have grown as candidates for biomarkers and vaccines. Proteins are vastly different with mass ranging from 1 kDa to well over 1 MDa and various types of post translational modifications. The structural heterogeneity that proteins can exhibit demonstrates the need for high resolution mass spectrometry methods. The combination of native mass spectrometry and soft ionization sources allow for preservation of structures seen in solution as analytes enter the gas phase. By developing methods that probe these structures, the information gathered can be related to the native structures in solution. Here I show, gas phase ion/ion reactions that can be utilized for location of salt bridge structures, gas-phase crosslinking of homo and heterodimer protein complexes, and mass determination of large (>800 kDa) protein complexes. These methods allow for greater control, faster data acquisition, and minimal sample preparation. These methods were developed on modified Sciex TripleTOF 5600 and 4000 QTRAP tandem mass spectrometers.</p>

Objective: The aim of this study was to investigate inherent in vitro permeability, metabolism, and cytotoxicity of idebenone - an active used to protect skin as an anti-aging agent -and compare it to idebenone linoleate. Methods: Idebenone and idebenone linoleate were investigated in pig ear skin and melanoma (B16: F10 mouse) cells. Diffusion experiments were conducted at 37 °C (bath temperature) using Franz diffusion cells. Authentic metabolite samples were synthetically prepared. Samples were analyzed using liquid chromatography-mass spectrometry/mass spectrometry. Cell viability was determined via the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: Idebenone was shown to permeate across viable porcine ear tissue; there was no evidence that idebenone linoleate permeated across porcine ear tissue after 4 h. Idebenone was metabolized to idebenone acid in both pig ear and mouse melanocytes; only minor idebenone linoleate metabolism was observed. Idebenone displayed delayed in vitro toxicity (via MTT assay) in melanocytes, while idebenone linoleate displayed no such in vitro toxicity. Conclusions: The in vitro metabolism and cytotoxicity results suggest that metabolic activation of idebenone is the likely culprit that activates the skin irritation mechanism via idebenone in vivo usage. An idebenone ester (e.g. idebenone linoleate) appears to provide a superior in vitro safety profile over idebenone.

Raloxifene is a highly insoluble, highly metabolized serum estrogen receptor modulator approved for use in the treatment of osteoporosis. Hydroxybutenyl-beta-cyclodextrin (HBenBCD) is a novel solubility enhancer previously demonstrated to increase the oral bioavailability of tamoxifen, letrozole, and itraconazole. The current study evaluated the pharmacokinetics of raloxifene in oral and intravenous formulations with HBenBCD in male Wistar-Hannover rats. Analytical methodology to measure raloxifene and its metabolites was developed by measuring raloxifene metabolism in vitro. Formulation with HBenBCD significantly increased raloxifene oral bioavailability. Mean ± S.D. oral bioavailabilities were 2.6 ± 0.4% for raloxifene formulated with microcrystalline cellulose, 7.7 ± 2.1% for a solid capsule formulation of raloxifene:HBenBCD complex, and 5.7 ± 1.3% for a liquid-filled capsule formulation containing raloxifene:HBenBCD/PEG400/H2O. Relative to raloxifene/microcrystalline filled capsules, the presence of HBenBCD in the solid capsule formulation afforded: (i) a decrease in raloxifene Tmax (2.5 ± 0.5 h versus 4.0 ± 0.5 h); (ii) a two-fold increase in raloxifene Cmax and a three-fold increase in raloxifene AUC; and (iii) a 12-fold increase in raloxifene glucuronide Cmax and a 6.5-fold increase in raloxifene glucuronide AUC. Hence, these studies demonstrate that raloxifene formulations containing HBenBCD significantly increased the oral bioavailability in rats relative to formulations that did not contain HBenBCD.

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The ability to identify and confirm a compound using mass spectrometry usually involves time consuming sample preparation and method development. The open-air ion source DART (Direct Analysis in Real Time) can ionize compounds in the gas, solid, or liquid phase without chromatography or sample preparation due to the interactions of helium metastable atoms with gas molecules commonly found in air. The coupling of the DART to a time-of-flight (TOF) mass spectrometer allows the rapid determination of an analyte's elemental composition based on accurate mass measurement and isotope peak intensities. Mass spectrometric fragmentation data can aid in the structural identification of an analyte as compounds produce characteristic fragment-ions based on their structure. The TOP's ability to produce fragmentation spectra was compared to the more traditional tandem mass spectral method (MS/MS) considering the TOF lacks the ability to select pre-cursor ions. The TOF produced in-source CAD (collisionally activated dissociation) spectra comparable to MS/MS spectra for three well known pharmaceuticals acetaminophen, phenylbutazone and clenbuterol. Further structural confirmation was explored through a determination of the number of active hydrogen atoms in an analyte molecule achieved by hydrogen/deuterium (H/D) exchange by treatment with deuterium oxide (D20) in the DART sample gap. Mass spectra acquired in the presence of D20 of analytes containing active hydrogen atoms associated with hydroxyl, amino and carboxylic acid groups showed that H/D exchange was predictable and reproducible. Using accurate mass measurement and isotope peak intensities, the elemental composition of an unknown captured on filter paper was identified as dipropylene glycol (DPG) analyzed directly from the surface of the filter paper. Data from in-source CAD and H/D exchange of both the unknown and authentic standards confirmed that the unknown was DPG. The cross-correlation of accurate mass measurement and isotope peak intensities, in-source CAD and HID exchange data provided an unambiguous identification of the contaminant melamine in dog food without the need for any sample preparation. Once analytes are identified and confirmed, quantitation of the analyte is desirable. The calibration curves here are constructed using the net extracted ion-current associated with the analyte relative to the internal standard. In cough syrup, a complicated matrix, the linearity, R2, is shown to be 0.992.

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Indiana University-Purdue University Indianapolis (IUPUI) / Human apurinic/apyrimidinic endonuclease 1 (hApe1) is a multi-functional protein having two major functions: apurinic/apyrimidinic endonuclease activity for DNA damage repair and redox activity for gene regulation. Many studies have shown the action of Ape1 in the base excision repair pathway leading to cell survival. It has also been reported that Ape1 reduces a number of important transcription factors that are involved in cancer promotion and progression. Though the repair activity is well understood, the redox mechanism is not yet clear. What is known about Ape1 is its structure and that it contains seven cysteines (C65, C93, C99, C138, C208, C296, and C310), none of which are disulfide bonded. Two of these cysteines, C99 and C138, are solvent-accessible, and C65, C93, and C99 are located in the redox domain. It is believed that one or more cysteines are involved in the redox function and is hypothesized that hApe1 reduces the down-stream transcription factors by a disulfide exchange mechanism. E3330, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquninoyl)]2-nonyl-2-propenoic acid, is a specific inhibitor for the redox function of hApe1. The interaction mechanism is not known. Using N-Ethylmaleimide (NEM) chemical footprinting, combined with Hydrogen/Deuterium Exchange (HDX) data, we propose that a locally unfolded form coexists with the folded form in an equilibrium that is driven by irreversible NEM labeling, and that E3330 interacts with and stabilizes this locally unfolded form. This locally unfolded form is thereby proposed to be the redox-active form. We further support this claim with LC-MS/MS analysis showing an increase of disulfide bonds induced by E3330 among the cysteines in the redox domain, which would be too far apart from each other in the folded form to form a disulfide bond. We also studied three analogs of E3330. The need for an E3330 analog is to develop a more efficient and effective compound that would allow for sub-micromolar levels of activity (E3330 requires a micromolar amount). Study of the analogs will also allow us to gain perspective of the mechanism or mechanisms of E3330’s activity in Ape1’s redox function.