Hydrogen Peroxide Modifies Aß-Membrane Interactions with Implications for Aß40 Aggregation.

Alzheimer's disease (AD) is pathologically characterized by the formation of extracellular senile plaques, predominately comprised of aggregated ß-amyloid (Aß), deposited in the brain. Aß aggregation can result in a myriad of distinct aggregate species, from soluble oligomers to insoluble fibrils. Aß strongly interacts with membranes, which can be linked to a variety of potential toxic mechanisms associated with AD. Oxidative damage accompanies the formation of Aß aggregates, with a 10-50% proportion of Aß aggregates being oxidized in vivo. Hydrogen peroxide (H2O2) is a reactive oxygen species implicated in a number of neurodegenerative diseases. Recent evidence has demonstrated that the H2O2 concentration fluctuates rapidly in the brain, resulting in large concentration spikes, especially in the synaptic cleft. Here, the impact of environmental H2O2 on Aß aggregation in the presence and absence of lipid membranes is investigated. Aß40 was exposed to H2O2, resulting in the selective oxidation of methionine 35 (Met35) to produce Aß40Met35[O]. While oxidation mildly reduced the rate of Aß aggregation and produced a distinct fibril morphology at high H2O2 concentrations, H2O2 had a much more pronounced impact on Aß aggregation in the presence of total brain lipid extract vesicles. The impact of H2O2 on Aß aggregation in the presence of lipids was associated with a reduced affinity of Aß for the vesicle surface. However, this reduced vesicle affinity was predominately associated with lipid peroxidation rather than Aß oxidation.

Regioselective Copper-Catalyzed Boracarboxylation of Vinyl Arenes.

Regioselective copper-catalyzed boracarboxylation of vinyl arenes with bis(pinacolato)diboron and carbon dioxide has been achieved. New boron-functionalized α-aryl carboxylic acids, including nonsteroidal anti-inflammatory drugs (NSAIDs), are obtained in moderate to excellent yields. The synthetic utility of the transformation was shown through subsequent derivatization of the carbon-boron bond yielding formal hydroxy- and fluorocarboxylation products as well as anionic difluoroboralactones.

Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 2. Assessing Charge Site Location and Isotope Scrambling.

Ion mobility spectrometry (IMS) coupled with gas-phase hydrogen deuterium exchange (HDX)-mass spectrometry (MS) and molecular dynamic simulations (MDS) has been used for structural investigation of anions produced by electrospraying a sample containing a synthetic peptide having the sequence KKDDDDDIIKIIK. In these experiments the potential of the analytical method for locating charge sites on ions as well as for utilizing collision-induced dissociation (CID) to reveal the degree of deuterium uptake within specific amino acid residues has been assessed. For diffuse (i.e., more elongated) [M - 2H](2-) ions, decreased deuterium content along with MDS data suggest that the D4 and D6 residues are charge sites, whereas for the more diffuse [M - 3H](3-) ions, the data suggest that the D4, D7, and the C-terminus are deprotonated. Fragmentation of mobility-selected, diffuse [M - 2H](2-) ions to determine deuterium uptake at individual amino acid residues reveals a degree of deuterium retention at incorporation sites. Although the diffuse [M - 3H](3-) ions may show more HD scrambling, it is not possible to clearly distinguish HD scrambling from the expected deuterium uptake based on a hydrogen accessibility model. The capability of the IMS-HDX-MS/MS approach to provide relevant details about ion structure is discussed. Additionally, the ability to extend the approach for locating protonation sites on positively-charged ions is presented.

Comparative plasma proteomic studies of pulmonary TiO2 nanoparticle exposure in rats using liquid chromatography tandem mass spectrometry.

Mounting evidence suggests that pulmonary exposure to nanoparticles (NPs) has a toxic effect on biological systems. A number of studies have shown that exposure to NPs result in systemic inflammatory response, oxidative stress, and leukocyte adhesion. However, significant knowledge gaps exist for understanding the key molecular mechanisms responsible for altered microvasculature function. Utilizing comprehensive LC-MS/MS and comparative proteomic analysis strategies, important proteins related to TiO2 NP exposure in rat plasma have been identified. Molecular pathway analysis of these proteins revealed 13 canonical pathways as being significant (p ≤ 0.05), but none were found to be significantly up or down-regulated (z>|2|). This work lays the foundation for future research that will monitor relative changes in protein abundance in plasma and tissue as a function of post-exposure time and TiO2 NP dosage to further elucidate mechanisms of pathway activation as well as to decipher other affected pathways.

Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 3. Estimating Surface Area Exposure by Deuterium Uptake.

Gas-phase hydrogen deuterium exchange (HDX), collision cross section (CCS) measurement, and molecular dynamics simulation (MDS) techniques were utilized to develop and compare three methods for estimating the relative surface area exposure of separate peptide chains within bovine insulin ions. Electrosprayed [M - 3H](3-) and [M - 5H](5-) insulin ions produced a single conformer type with respective collision cross sections of 528 ± 5 Å(2) and 808 ± 2 Å(2). [M - 4H](4-) ions were comprised of more compact (Ω = 676 ± 3 Å(2)) and diffuse (i.e., more elongated, Ω = 779 ± 3 Å(2)) ion conformer types. Ions were subjected to HDX in the drift tube using D2O as the reagent gas. Collision-induced dissociation was used to fragment mobility-selected, isotopically labeled [M - 4H](4-) and [M - 5H](5-) ions into the protein subchains. Deuterium uptake levels of each chain can be explained by limited inter-chain isotopic scrambling upon collisional activation. Using nominal ion structures from MDS and a hydrogen accessibility model, the deuterium uptake for each chain was correlated to its exposed surface area. In separate experiments, the per-residue deuterium content for the protonated and deprotonated ions of the synthetic peptide KKDDDDDIIKIIK were compared. The differences in deuterium content indicated the regional HDX accessibility for cations versus anions. Using ions of similar conformational type, this comparison highlights the complementary nature of HDX data obtained from positive- and negative-ion analysis.

Advances in ion mobility-mass spectrometry instrumentation and techniques for characterizing structural heterogeneity.

Over the last decade, the field of ion mobility-mass spectrometry (IM-MS) has experienced dramatic growth in its application toward ion structure characterization. Enabling advances in instrumentation during this time period include improved conformation resolution and ion sensitivity. Such advances have rendered IM-MS a powerful approach for characterizing samples presenting a diverse array of ion structures. The structural heterogeneity that can be interrogated by IM-MS techniques now ranges from samples containing mixtures of small molecules exhibiting a variety of structural types to those containing very large protein complexes and subcomplexes. In addition to this diversity, IM-MS techniques have been used to probe spontaneous and induced structural transformations occurring in solution or the gas phase. To support these measurement efforts, significant advances have been made in theoretical methods aimed at translating IM-MS data into structural information. These efforts have ranged from providing more reliable trial structures for comparison to the experimental measurements to dramatically reducing the time required to calculate collision cross sections for such structures. In this short review, recent advances in developments in IM-MS instrumentation, techniques, and theory are discussed with regard to their implications for characterization of gas- and solution-phase structural heterogeneity.

Online deuterium hydrogen exchange and protein digestion coupled with ion mobility spectrometry and tandem mass spectrometry.

Online deuterium hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within ∼60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment. Extracted ion drift time distributions (XIDTDs) of deuterated peptic peptides are mobility-matched to corresponding XIDTDs of undeuterated peptic peptides that were identified using collision-induced dissociation (CID). Matching XIDTDs allows a straightforward identification and deuterium retention evaluation for labeled peptides. Aside from the mobility separation, the ion trapping capabilities of the LTQ, combined with ETD, are demonstrated to provide single-residue resolution. Deuterium retention for the c- series ions across residues M(1)-L(15) and N(25)-R(42) are in good agreement with the known secondary structural elements within ubiquitin.

Ion mobility spectrometry-hydrogen deuterium exchange mass spectrometry of anions: part 1. Peptides to proteins.

Ion mobility spectrometry (IMS) coupled with hydrogen deuterium exchange (HDX)-mass spectrometry (MS) has been used to study the conformations of negatively-charged peptide and protein ions. Results are presented for ion conformers of angiotensin 1, a synthetic peptide (SP), bovine insulin, ubiquitin, and equine cytochrome c. In general, the SP ion conformers demonstrate a greater level of HDX efficiency as a greater proportion of the sites undergo HDX. Additionally, these ions exhibit the fastest rates of exchange. Comparatively, the angiotensin 1 ions exhibit a lower rate of exchange and HDX level presumably because of decreased accessibility of exchange sites by charge sites. The latter are likely confined to the peptide termini. Insulin ions show dramatically reduced HDX levels and exchange rates, which can be attributed to decreased conformational flexibility resulting from the disulfide bonds. For the larger ubiquitin and protein ions, increased HDX is observed for larger ions of higher charge state. For ubiquitin, a conformational transition from compact to more elongated species (from lower to higher charge states) is reflected by an increase in HDX levels. These results can be explained by a combination of interior site protection by compact conformers as well as decreased access by charge sites. The elongated cytochrome c ions provide the largest HDX levels where higher values correlate with charge state. These results are consistent with increased exchange site accessibility by additional charge sites. The data from these enhanced IMS-HDX experiments are described in terms of charge site location, conformer rigidity, and interior site protection.

Combining ion mobility spectrometry with hydrogen-deuterium exchange and top-down MS for peptide ion structure analysis.

The gas-phase conformations of electrosprayed ions of the model peptide KKDDDDIIKIIK have been examined by ion mobility spectrometry (IMS) and hydrogen deuterium exchange (HDX)-tandem mass spectrometry (MS/MS) techniques. [M+4H](4+) ions exhibit two conformers with collision cross sections of 418 Å(2) and 471 Å(2). [M+3H](3+) ions exhibit a predominant conformer with a collision cross section of 340 Å(2) as well as an unresolved conformer (shoulder) with a collision cross section of ~367 Å(2). Maximum HDX levels for the more compact [M+4H](4+) ions and the compact and partially-folded [M+3H](3+) ions are ~12.9, ~15.5, and ~14.9, respectively. Ion structures obtained from molecular dynamics simulations (MDS) suggest that this ordering of HDX level results from increased charge-site/exchange-site density for the more compact ions of lower charge. Additionally, a new model that includes two distance calculations (charge site to carbonyl group and carbonyl group to exchange site) for the computer-generated structures is shown to better correlate to the experimentally determined per-residue deuterium uptake. Future comparisons of IMS-HDX-MS data with structures obtained from MDS are discussed with respect to novel experiments that will reveal the HDX rates of individual residues.

A new ion mobility-linear ion trap instrument for complex mixture analysis.

A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility. Even though the gas-phase separation and the mass measurement steps are each conducted in an ion filtering mode, detection limits for mobility-selected peptide ions are in the tens of attomole range. In addition to ion separation, the low-pressure drift tube can be used as an ion fragmentation cell yielding mobility-resolved fragment ions that can be subsequently analyzed by multistage tandem mass spectrometry (MS(n)) methods in the ion trap. Because of the ion trap configuration, these methods can be comprised of any number (limited by ion signal) of collision-induced dissociation (CID) and electron transfer dissociation (ETD) processes. The high reproducibility of the gas-phase separation allows for comparison of two-dimensional ion mobility spectrometry (IMS)-MS data sets in a pixel-by-pixel fashion without the need for data set alignment. These advantages are presented in model analyses representing mixtures encountered in proteomics and metabolomics experiments.