Allison S. Danell

FRET measurements of trapped oligonucleotide duplexes

Sensitive methods developed to measure laser-induced fluorescence from trapped ions have been applied to study the intermediate states of the dissociation of oligonucleotide duplexes. Double-stranded oligonucleotide anions were prepared with FRET donor/acceptor pairs attached. Gas phase ions were generated by electrospray ionization and injected into a heated quadrupole ion trap where they were stored and exposed to Nd:YAG laser pulses at 532 nm. The dissociation of the duplexes into single strands was monitored by both fluorescence and mass spectra. The comparison of the degree of dissociation of the duplex observed in the mass spectra to the fluorescence intensity of the donor allows for the determination of the intermediate conformations of the unzipping duplex. The observation of changes in the donor fluorescence in the absence of single strands correlates with an intermediate state characterized by threshold unzipping at the weaker binding end of the duplex. These data are consistent with a model of intermediate state formation as a function of temperature. These studies suggest that FRET methods will be useful to obtain direct measurements, which characterize the dynamics of conformational changes of biomolecules in the gas phase.

The use of static pressures of heavy gases within a quadrupole ion trap.

The performance of quadrupole ion traps using argon or air as the buffer gas was evaluated and compared to the standard helium only operation. In all cases a pure buffer gas, not mixtures of gases, was investigated. Experiments were performed on a Bruker Esquire ion trap, a Finnigan LCQ, and a Finnigan ITMS for comparison. The heavier gases were found to have some advantages, particularly in the areas of sensitivity and collision-induced dissociation efficiency; however, there is a significant resolution loss due to dissociation and/or scattering of ions. Additionally, the heavier gases were found to affect ion activation and deactivation during MS/MS, influencing the product ion intensities observed. Finally, the specific quadrupole ion trap design and the ion ejection parameters were found to be crucial in the quality of the spectra obtained in the presence of heavy gases. Operation with static pressures of heavy gases can be beneficial under certain design and operating conditions of the quadrupole ion trap.

Fraying and electron autodetachment dynamics of trapped gas phase oligonucleotides.

Sensitive methods recently developed to measure laser-induced fluorescence from trapped ions have been applied to study the dynamics of double- and single-stranded oligonucleotides. In this paper, the fraying of duplex terminal base pairs has been identified by measuring the donor fluorescence as a function of temperature from an oligonucleotide duplex labeled with a pair of FRET dyes. Comparison of the degree of dissociation of 14-mer duplexes observed in the mass spectra with the fluorescence intensity of the donor enables intermediate conformations of the unzipping duplex at the weaker binding end of the duplex to be identified. The autodetachment of electrons from double- and single-stranded oligonucleotide anions has been observed in a gas phase environment. To characterize this process, measurements were performed on 7-mers prepared without FRET fluorophores attached. The dependence of the decay rates of trapped anions have been measured as a function of charge state and temperature for various base compositions. An exceptionally strong dependence of the decay rate on base composition has been identified. The physical basis for this process will be discussed.

The transcriptional regulator Np20 is the zinc uptake regulator in Pseudomonas aeruginosa.

Zinc is essential for all bacteria, but excess amounts of the metal can have toxic effects. To address this, bacteria have developed tightly regulated zinc uptake systems, such as the ZnuABC zinc transporter which is regulated by the Fur-like zinc uptake regulator (Zur). In Pseudomonas aeruginosa, a Zur protein has yet to be identified experimentally, however, sequence alignment revealed that the zinc-responsive transcriptional regulator Np20, encoded by np20 (PA5499), shares high sequence identity with Zur found in other bacteria. In this study, we set out to determine whether Np20 was functioning as Zur in P. aeruginosa. Using RT-PCR, we determined that np20 (hereafter known as zur) formed a polycistronic operon with znuC and znuB. Mutant strains, lacking the putative znuA, znuB, or znuC genes were found to grow poorly in zinc deplete conditions as compared to wild-type strain PAO1. Intracellular zinc concentrations in strain PAO-Zur (Δzur) were found to be higher than those for strain PAO1, further implicating the zur as the zinc uptake regulator. Reporter gene fusions and real time RT-PCR revealed that transcription of znuA was repressed in a zinc-dependent manner in strain PAO1, however zinc-dependent transcriptional repression was alleviated in strain PAO-Zur, suggesting that the P. aeruginosa Zur homolog (ZurPA) directly regulates expression of znuA. Electrophoretic mobility shift assays also revealed that recombinant ZurPA specifically binds to the promoter region of znuA and does not bind in the presence of the zinc chelator N,N′,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN). Taken together, these data support the notion that Np20 is the P. aeruginosa Zur, which regulates the transcription of the genes encoding the high affinity ZnuABC zinc transport system.

Charge permutation reactions in beam type mass spectrometers

Abstract This is a review of the gas-phase reactions, in beam type mass spectrometers, that change the charge states of ions. The Cooks research group has been a leader in both the understanding and use of these charge-changing reactions. The charge of ions can be manipulated in beam type instruments via collisions with neutral gas atoms or molecules in the high ion kinetic energy regime. The two major processes, charge inversion and charge stripping due to high energy ion/neutral collisions, are discussed. Charge permutation reactions often result in the formation of unique ion structures that aid in the differentiation of isomers. The products of charge permutation reactions may possess excess internal energy that cause the ions to fragment, and these fragment ions may provide complementary information to that obtained from high energy collision-induced dissociation. The charge of ions also can be changed as a result of a collision with a surface in the low ion kinetic energy regime. Charge exchange and charge inversion processes that occur as a result of low energy ion/surface collisions are presented.

Arachidonoyl‐ethanolamide activates endoplasmic reticulum stress‐apoptosis in tumorigenic keratinocytes: Role of cyclooxygenase‐2 and novel J‐series prostamides

Non‐melanoma skin cancer and other epithelial tumors overexpress cyclooxygenase‐2 (COX‐2), differentiating them from normal cells. COX‐2 metabolizes arachidonic acid to prostaglandins including, the J‐series prostaglandins, which induce apoptosis by mechanisms including endoplasmic reticulum (ER) stress. Arachidonoyl‐ethanolamide (AEA) is a cannabinoid that causes apoptosis in diverse tumor types. Previous studies from our group demonstrated that AEA was metabolized by COX‐2 to J‐series prostaglandins. Thus, the current study examines the role of COX‐2, J‐series prostaglandins, and ER stress in AEA‐induced apoptosis. In tumorigenic keratinocytes that overexpress COX‐2, AEA activated the PKR‐like ER kinase (PERK), inositol requiring kinase‐1 (IRE1), and activating transcription factor‐6 (ATF6) ER stress pathways and the ER stress apoptosis‐associated proteins, C/EBP homologous protein‐10 (CHOP10), caspase‐12, and caspase‐3. Using an ER stress inhibitor, it was determined that ER stress was required for AEA‐induced apoptosis. To evaluate the role of COX‐2 in ER stress‐apoptosis, HaCaT keratinocytes with low endogenous COX‐2 expression were transfected with COX‐2 cDNA or an empty vector and AEA‐induced ER stress‐apoptosis occurred only in the presence of COX‐2. Moreover, LC‐MS analysis showed that the novel prostaglandins, 15‐deoxyΔ12,14PGJ2‐EA and Δ12PGJ2/PGJ2‐EA, were synthesized from AEA. These findings suggest that AEA will be selectively toxic in tumor cells that overexpress COX‐2 due to the metabolism of AEA by COX‐2 to J‐series prostaglandin‐ethanolamides (prostamides). Hence, AEA may be an ideal topical agent for the elimination of malignancies that overexpress COX‐2.

Zinc Deposition During ESI-MS Analysis of Peptide-Zinc Complexes

Electrospray ionization (ESI) mass spectrometry (MS) has proven to be an extremely powerful technique for studying the stoichiometry and binding strength of peptide-metal complexes. We have found a significant new problem in the ESI-MS of zinc-peptide systems involving the deposition of zinc in the ESI emitter. This deposition of zinc during the experiment removes a significant amount of zinc ions from the solution, impacting the resulting mass spectral intensities used to quantify the amount of the zinc-bound species. Analysis of infused zinc-peptide samples with atomic absorption spectrometry and with a custom-built nanoflow ESI source confirms the alteration of the analyte solutions with positive or negative or no potential applied to the emitter. Ultimately, the location of the zinc deposition was determined to be the stainless steel emitter. The use of a custom-built nanoESI interface using glass emitters was found to mitigate the zinc deposition problem. The phenomenon of metal deposition warrants further investigation as it may not be limited to just zinc and may represent a significant obstacle in the ESI-MS analysis of all protein-metal systems.

Evidence for ionization-related conformational differences of peptide ions in a quadrupole ion trap

The differences in boundary-activated dissociation (BAD) onsets have been investigated for peptide ions that were generated by two different ionization techniques, nanoflow electros-pray ionization (nanoESI) and liquid secondary-ion mass spectrometry (LSIMS). BAD onsets of these ions were determined to compare the relative internal energies of the ions. Protonated peptide ions formed by nanoESI had lower BAD onsets than ions formed by LSIMS. The BAD onsets of peptides derivatized to have a fixed charge on the N-terminus also were lower for those generated by nanoESI than those generated by LSIMS. The BAD onsets of ions formed by nanoESI did not change with the variation of collisional cooling periods after gating ions into the ion trap and after isolating them prior to dissociation, indicating that the ions formed by the two ionization techniques would not adopt the same energy distributions. It is proposed that the ions formed by the two techniques differ in secondary structure, and the LSIMS ions are collisionally cooled to a lower local minimum along the potential energy surface than the nanoESI ions. Ions formed by both techniques show the same dissociation patterns, so assuming the absolute energy required for dissociation is the same, the LSIMS ions possess a higher critical energy of dissociation. This leads to the observation of the higher BAD onset.

Characterization of perfluoroalkyl acrylic oligomers by electrospray ionization time‐of‐flight mass spectrometry

Electrospray ionization time-of-flight mass spectrometry (ESI-ToF-MS) has been successfully employed for the characterization of molecular weight, molecular weight distribution and end groups for bromine-terminated perfluoroalkyl acrylate oligomers prepared using atom transfer radical polymerization. Intact oligomers and smaller quantities of common side products were observed from m/z 1000 to 4000 cationized with a sodium ion, a difluorobenzyl cation or a proton with a minimum of multiply charged species. Number average molecular weight and weight average molecular weight for both the samples that were characterized were in reasonable agreement with independent measurements conducted using GPC-MALS and (1)H NMR spectroscopy.

Note: A simple dual polarity dual nanoelectrospray ionization source for ion/ion reactions

A continuously operated dual polarity dual nanoelectrospray ionization source has been constructed and tested. A commercial quadrupole ion trap mass spectrometer was modified to accumulate and trap ions of opposite charge. All changes to the commercial three-dimensional quadrupole ion trap have been made external to the instrument outside of the vacuum system. Few hardware modifications were required because the two emitters send ion beams through the same transmission guides. Computer controlled source voltage polarities are switched quickly and efficiently to transmit one of two continuously generated ion beams. With customized software, this design has proved simple to implement and to operate.