A standardized method for plasma extracellular vesicle isolation and size distribution analysis.

The following protocol describes our workflow for isolation and quantification of plasma extracellular vesicles (EVs). It requires limited sample volume so that the scientific value of specimens is maximized. These steps include isolation of vesicles by automated size exclusion chromatography and quantification by tunable resistive pulse sensing. This workflow optimizes reproducibility by minimizing variations in processing, handling, and storage of EVs. EVs have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. This standardized protocol is scalable and ensures efficient recovery of physiologically intact EVs that may be used in a variety of downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely. Absolute quantification corrects for variations in EV number and size, offering a novel method of standardization in downstream applications.

Conformer-Dependent Chemistry: Experimental Product Branching of the Vinyl Alcohol + OH + O2 Reaction.

The concentration of formic acid in Earth's troposphere is underestimated by detailed chemical models compared to field observations. Phototautomerization of acetaldehyde to its less stable tautomer vinyl alcohol, followed by the OH-initiated oxidation of vinyl alcohol, has been proposed as a missing source of formic acid that improves the agreement between models and field measurements. Theoretical investigations of the OH + vinyl alcohol reaction in excess O2 conclude that OH addition to the α carbon of vinyl alcohol produces formaldehyde + formic acid + OH, whereas OH addition to the ß site leads to glycoaldehyde + HO2. Furthermore, these studies predict that the conformeric structure of vinyl alcohol controls the reaction pathway, with the anti-conformer of vinyl alcohol promoting α OH addition, whereas the syn-conformer promotes ß addition. However, the two theoretical studies reach different conclusions regarding which set of products dominate. We studied this reaction using time-resolved multiplexed photoionization mass spectrometry to quantify the product branching fractions. Our results, supported by a detailed kinetic model, conclude that the glycoaldehyde product channel (arising mostly from syn-vinyl alcohol) dominates over formic acid production with a 3.6:1.0 branching ratio. This result supports the conclusion of Lei et al. that conformer-dependent hydrogen bonding at the transition state for OH-addition controls the reaction outcome. As a result, tropospheric oxidation of vinyl alcohol creates less formic acid than recently thought, increasing again the discrepancy between models and field observations of Earth's formic acid budget.

Radical-Radical Reactions in Molecular Weight Growth: The Phenyl + Propargyl Reaction.

The mechanism for hydrocarbon ring growth in sooting environments is still the subject of considerable debate. The reaction of phenyl radical (C6H5) with propargyl radical (H2CCCH) provides an important prototype for radical-radical ring-growth pathways. We studied this reaction experimentally over the temperature range of 300-1000 K and pressure range of 4-10 Torr using time-resolved multiplexed photoionization mass spectrometry. We detect both the C9H8 and C9H7 + H product channels and report experimental isomer-resolved product branching fractions for the C9H8 product. We compare these experiments to theoretical kinetics predictions from a recently published study augmented by new calculations. These ab initio transition state theory-based master equation calculations employ high-quality potential energy surfaces, conventional transition state theory for the tight transition states, and direct CASPT2-based variable reaction coordinate transition state theory (VRC-TST) for the barrierless channels. At 300 K only the direct adducts from radical-radical addition are observed, with good agreement between experimental and theoretical branching fractions, supporting the VRC-TST calculations of the barrierless entrance channel. As the temperature is increased to 1000 K we observe two additional isomers, including indene, a two-ring polycyclic aromatic hydrocarbon, and a small amount of bimolecular products C9H7 + H. Our calculated branching fractions for the phenyl + propargyl reaction predict significantly less indene than observed experimentally. We present further calculations and experimental evidence that the most likely cause of this discrepancy is the contribution of H atom reactions, both H + indenyl (C9H7) recombination to indene and H-assisted isomerization that converts less stable C9H8 isomers into indene. Especially at low pressures typical of laboratory investigations, H-atom-assisted isomerization needs to be considered. Regardless, the experimental observation of indene demonstrates that the title reaction leads, either directly or indirectly, to the formation of the second ring in polycyclic aromatic hydrocarbons.

Dissociative Photoionization of Methyl Vinyl Ketone-Thermochemical Anchors and a Drifting Methyl Group.

The dissociative photoionization of methyl vinyl ketone (MVK), an important intermediate in the atmospheric oxidation of isoprene, has been studied by photoelectron photoion coincidence spectroscopy. In the photon energy range of 9.5-13.8 eV, four main fragment ions were detected at m/z 55, 43, 42, and 27 aside from the parent ion at m/z 70. The m/z 55 fragment ion (C2H3CO+) is formed from ionized MVK by direct methyl loss, while breaking the C-C bond on the other side of the carbonyl group results in the acetyl cation (CH3CO+, m/z 43) and the vinyl radical. The m/z 42 fragment ion is formed via a CO-loss from the molecular ion after a methyl shift. The lightest fragment ion, the vinyl cation (C2H3+ at m/z 27), is produced in two different reactions: acetyl radical loss from the molecular ion and CO-loss from C2H3CO+. Their contributions to the m/z 27 signal are quantified based on the acetyl and vinyl fragment thermochemical anchors and quantum chemical calculations. Based on the experimentally derived appearance energy of the m/z 43 fragment ion, a new, experimentally derived heat of formation is proposed herein for gaseous methyl vinyl ketone (ΔfH0K = -94.3 ± 4.8 kJ mol-1; ΔfH298K = -110.5 ± 4.8 kJ mol-1), together with cationic heats of formation and bond dissociation energies.

The Vagabond Fluorine Atom: Dissociative Photoionization of trans-1,3,3,3-Tetrafluoropropene.

The dissociative photoionization of trans-1,3,3,3-tetrafluoropropene (HFO-1234ze) was investigated by imaging photoelectron photoion coincidence (PEPICO) spectroscopy. From the threshold photoelectron spectrum (TPES), an adiabatic ionization energy of 10.91 ± 0.05 eV is determined and reported for the first time. Over a 4 eV wide range, internal-energy selected trans-1,3,3,3-tetrafluoropropene cations decay by three parallel dissociative photoionization channels, which were modeled using statistical theory. The 0 K appearance energies of CF2CHCF2 (H-loss, m/z 113), CFHCHCF2 (F-loss, m/z 95), and CH2═CF2 (CF2-loss, m/z 64) fragment ions were determined to be 12.247 ± 0.030, 12.66 ± 0.10, and 12.80 ± 0.05 eV, respectively. From the last, the heat of formation of neutral trans-1,3,3,3-tetrafluoropropene was determined to be -779.9 ± 9.7 kJ/mol. While the lowest-energy fluorine loss occurs directly, the first H-loss and CF2-loss channels involve both a fluorine- and a hydrogen-migration prior to dissociation. At higher internal energies, several other rearrangement pathways open up, which involve fluorine and hydrogen transfer and, through fluorine loss, lead to the formation of several additional isomeric allylic fragment ions.

Effects of vibrational excitation on the F + H2O → HF + OH reaction: dissociative photodetachment of overtone-excited [F-H-OH].

The reaction F + H2O → HF + OH is a four-atom system that provides an important benchmark for reaction dynamics. Hydrogen atom transfer at the transition state for this reaction is expected to exhibit a strong dependence on reactant vibrational excitation. In the present study, the vibrational effects are examined by photodetachment of vibrationally excited F-(H2O) precursor anions using photoelectron-photofragment coincidence (PPC) spectroscopy and compared with full six-dimensional quantum dynamical calculations on ab initio potential energy surfaces. Prior to photodetachment at hνUV = 4.80 eV, the overtone of the ionic hydrogen bond mode in the precursor F-(H2O), 2νIHB at 2885 cm-1, was excited using a tunable IR laser. Experiment and theory show that vibrational energy in the anion can be effectively carried away by the photoelectron upon a Franck-Condon photodetachment, and also show evidence for an increase of branching into the F + H2O reactant channel. The experimental results suggest a greater role for product rotational excitation than theory. Improved potential energy surfaces and longer wavepacket propagation times would be helpful to further examine the nature of the discrepancy.

Energetics and transition-state dynamics of the F + HOCH3 → HF + OCH3 reaction.

The F + HOCH3 → HF + OCH3 reaction is a system with 15 internal degrees of freedom that can provide a benchmark for the development of theory for increasingly complex chemical reactions. The dynamics of this reaction were studied by photoelectron-photofragment coincidence (PPC) spectroscopy carried out on the F-(HOCH3) anion, aided by a computational study of both the anion and neutral potential energy surfaces, with energies extrapolated to the CCSDT(Q)/CBS level of theory. Photodetachment at 4.80 eV accesses both the reactant and product channels for this reaction. In the product channel (HF + OCH3 + e-) of the neutral potential energy surface, vibrationally excited HF products in addition to the stable product-channel hydrogen-bonded complex (FH-OCH3) are observed in the PPC and photoelectron spectra. In addition, experimental evidence is observed for the reactant-channel van der Waals complex (F-HOCH3), in good agreement with the theoretical predictions. The relative stability of these long-lived complexes was probed by reducing the ion beam energy, increasing the product time-of-flight, indicating lifetimes on the microsecond timescale for the reactant- and product-channel complexes as well as providing evidence for long-lived vibrational Feshbach resonances associated with the HF(v > 0) + OCH3 product states. This system will provide a model for extending full-dimensionality quantum dynamics to larger numbers of degrees of freedom.

Imaging dynamics on the F + H2O -> HF + OH potential energy surfaces from wells to barriers.

The study of gas-phase reaction dynamics has advanced to a point where four-atom reactions are the proving ground for detailed comparisons between experiment and theory. Here, a combined experimental and theoretical study of the dissociation dynamics of the tetra-atomic FH2O system is presented, providing snapshots of the F + H2O → HF + OH reaction. Photoelectron-photofragment coincidence measurements of the dissociative photodetachment (DPD) of the F(-)(H2O) anion revealed various dissociation pathways along different electronic states. A distinct photoelectron spectrum of stable FH-OH complexes was also measured and attributed to long-lived Feshbach resonances. Comparison to full-dimensional quantum calculations confirms the sensitivity of the DPD measurements to the subtle dynamics on the low-lying FH2O potential energy surfaces over a wide range of nuclear configurations and energies.

Dissociative photodetachment of the ethoxide anion and stability of the ethoxy radical CH3CH2O•.

The ethoxy radical is an important species in combustion chemistry; however, considerable debate regarding the fragmentation pathways exists. In order to examine the stability and dissociation dynamics of the ethoxy radical in the two lowest electronic states, dissociative photodetachment experiments at 3.20 eV were carried out on the ethoxide anion, CH3CH2O(-), and its per-deuterated isotopologue. Production of excited radicals by photodetachment of the alkoxide anion was found to lead to only CH3 + H2CO products, with no indication of the energetically allowed H-loss channel, H + CH3CHO. Ab initio calculations for the anionic and neutral surfaces, including relevant isomerization and dissociation barriers, were carried out using the CBS-QB3 method to aid in interpretation of the data. The energetics observed in the photoelectron-photofragment coincidence spectra indicate that the calculated barrier (0.70 eV) for the process CH3CH2O → CH3 + H2CO and the stability of the CH3CH2O radical relative to those products are upper limits.

Synchrotron photoionization measurements of OH-initiated cyclohexene oxidation: ring-preserving products in OH + cyclohexene and hydroxycyclohexyl + O2 reactions.

Earlier synchrotron photoionization mass spectrometry experiments suggested a prominent ring-opening channel in the OH-initiated oxidation of cyclohexene, based on comparison of product photoionization spectra with calculated spectra of possible isomers. The present work re-examines the OH + cyclohexene reaction, measuring the isomeric products of OH-initiated oxidation of partially and fully deuterated cyclohexene. In particular, the directly measured photoionization spectrum of 2-cyclohexen-1-ol differs substantially from the previously calculated Franck-Condon envelope, and the product spectrum can be fit with no contribution from ring-opening. Measurements of H(2)O(2) photolysis in the presence of C(6)D(10) establish that the addition-elimination product incorporates the hydrogen atom from the hydroxyl radical reactant and loses a hydrogen (a D atom in this case) from the ring. Investigation of OH + cyclohexene-4,4,5,5-d(4) confirms this result and allows mass discrimination of different abstraction pathways. Products of 2-hydroxycyclohexyl-d(10) reaction with O(2) are observed upon adding a large excess of O(2) to the OH + C(6)D(10) system.