Pulse Duration Effects on Solution-Phase Protein Desorption in Laser Electrospray Mass Spectrometry.

The effect of laser pulse duration on the ablation of aqueous myoglobin is investigated using laser electrospray mass spectrometry (LEMS). Pulse durations of 55 femtoseconds (fs), 56 piscoseconds (ps), and 10 nanoseconds (ns) were used to ablate aqueous myoglobin from stainless-steel and quartz substrates. The integrated signal intensity of myoglobin increases with decreasing pulse duration for both substrates. Laser-induced thermal effects are assessed by the relative amount of solvent adduction and number of phosphate moieties adducted to myoglobin by each laser pulse duration. The mass spectra for 55 fs vaporization shows myoglobin with appreciable solvent and phosphate adduction and baseline elevation. The mass spectra for 10 ns ablation have minimal adduction and limited baseline elevation. Heat-induced conformation changes in myoglobin were used to measure the amount of thermal energy deposited by each laser pulse duration. Ablation using the 55 fs pulse revealed the highest ratio of unfolded to folded myoglobin in comparison to the 56 ps and 10 ns measurements due to increased droplet lifetime and consequent interaction with the acid in the electrospray solvent. Collisional activation and heated capillary temperature were employed to reduce the droplet lifetime and demonstrate that fs ablation preserves approximately 2 times more myoglobin folded conformation in comparison to ps and ns pulses.

Spectral Signatures of Ground- and Excited-State Wavepacket Interference after Impulsive Excitation.

Impulsive transient absorption spectroscopy is used to track the formation and evolution of vibrational coherences in cresyl violet perchlorate under different excitation conditions. Resonant and off-resonant pump pulses result in the selective formation of excited (S1)- and ground (S0)-state wavepackets. Partially resonant and broadband excitation conditions lead to the simultaneous formation of wavepackets in the ground and excited states. The wavepackets are characterized by the phase-flips in the coherent signal associated with wavepacket motion across the absorption and emission maxima and by a red shift of 2-10 cm-1 in the Raman features of the excited state compared to the ground-state wavepacket. We observe that, when wavepackets are simultaneously excited on the ground- and excited-state surfaces, interference on a picosecond timescale between coherent oscillations in the two wavepackets gives rise to features that cannot be attributed to the passage of a wavepacket through a conical intersection, such as shifting phase-flips and zero-amplitude nodes. Wavepacket filtering using windowed Fourier transforms highlights these interference effects and demonstrates that special care must be taken in order to properly interpret data that have been processed in this manner.

Formation of Copper(I) Oxide- and Copper(I) Cyanide-Polyacetonitrile Nanocomposites through Strong-Field Laser Processing of Acetonitrile Solutions of Copper(II) Acetate Dimer.

Irradiation studies of acetonitrile solutions of copper(II) acetate dimer ([Cu(OAc)2]2) using high energy, simultaneously spatially and temporally focused (SSTF) ultrashort laser pulses are reported. Under ambient conditions, irradiation for relatively short periods of time (10-20 s) selectively produces relatively small, narrowly size-dispersed (3.5 ± 0.7 nm) copper(I) oxide nanoparticles (Cu2O NPs) embedded in CuCN-polyacetonitrile polymers generated in situ by the laser. The Cu2O NPs become embedded in a CuCN-polyacetonitrile network as they form, stabilizing them and protecting the air-sensitive material from oxygen. Laser irradiation of acetonitrile causes fragmentation into transient radicals that initiate and terminate polymerization of acetonitrile. Control and mechanistic investigations reveal that HCN formed during laser irradiation reacts rapidly to reduce the Cu(II) centers in [Cu(OAc)2]2, leading to the formation of CuCN or, in the presence of water, Cu2O nanoparticles that bind and cross-link CuCN-polyacetonitrile chains. The acetate-bridged Cu(II) dimer unit is a required structural feature that functions to preorganize and direct the Cu(II) reduction and selective formation of CuCN and Cu2O nanoparticles. This study illustrates how rapid deposition of energy using shaped, ultrashort laser pulses can initiate multiple photolytic and thermal processes that lead to the selective formation of composite nanoparticle/polymer materials for applications in electronics and catalysis.

Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry.

Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (Kd) for hen egg white lysozyme (HEWL) and N,N',N″-triacetylchitotriose (NAG3) revealed an apparent Kd value of 313.2 ± 25.9 µM for the ligand titration method. Similar measurements for N,N',N″,N″'-tetraacetylchitotetraose (NAG4) revealed an apparent Kd of 249.3 ± 13.6 µM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a Kd value of 9.8 ± 0.6 µM. In a second LEMS approach, a calibrated measurement was used to determine a Kd value of 6.8 ± 1.5 µM for NAG3. The capture efficiency of LEMS was measured to be 3.6 ± 1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted Kd value was 11.3 µM for NAG3 and 9.0 µM for NAG4. The calibration method for measuring Kd developed in this study can be applied to solutions containing unknown analyte concentrations. Graphical Abstract.

Direct Analysis of Proteins from Solutions with High Salt Concentration Using Laser Electrospray Mass Spectrometry.

The detection of lysozyme, or a mixture of lysozyme, cytochrome c, and myoglobin, from solutions with varying salt concentrations (0.1 to 250 mM NaCl) is compared using laser electrospray mass spectrometry (LEMS) and electrospray ionization-mass spectrometry (ESI-MS). Protonated protein peaks were observed up to a concentration of 250 mM NaCl in the case of LEMS. In the case of ESI-MS, a protein solution with salt concentration > 0.5 mM resulted in predominantly salt-adducted features, with suppression of the protonated protein ions. The constituents in the mixture of proteins were assignable up to 250 mM NaCl for LEMS and were not assignable above a NaCl concentration of 0.5 mM for ESI. The average sodium adducts (< n >) bound to the 7+ charge state of lysozyme for LEMS measurements from salt concentrations of 2.5, 25, 50, and 100 mM NaCl are 1.71, 5.23, 5.26, and 5.11, respectively. The conventional electrospray measurements for lysozyme solution containing salt concentrations of 0.1, 1, 2, and 5 mM NaCl resulted in < n > of 2.65, 6.44, 7.57, and 8.48, respectively. LEMS displays an approximately two orders of magnitude higher salt tolerance in comparison with conventional ESI-MS. The non-equilibrium partitioning of proteins on the surface of the charged droplets is proposed as the mechanism for the high salt tolerance phenomena observed in the LEMS measurements. Graphical Abstract ᅟ.

Conserving Coherence and Storing Energy during Internal Conversion: Photoinduced Dynamics of cis- and trans-Azobenzene Radical Cations.

Light harvesting via energy storage in azobenzene has been a key topic for decades and the process of energy distribution over the molecular degrees of freedom following photoexcitation remains to be understood. Dynamics of a photoexcited system can exhibit high degrees of nonergodicity when it is driven by just a few degrees of freedom. Typically, an internal conversion leads to the loss of such localization of dynamics as the intramolecular energy becomes statistically redistributed over all molecular degrees of freedom. Here, we present a unique case where the excitation energy remains localized even subsequent to internal conversion. Strong-field ionization is used to prepare cis- and trans-azobenzene radical cations on the D1 surface with little excess energy at the equilibrium neutral geometry. These D1 ions are preferably formed because in this case D1 and D0 switch place in the presence of the strong laser field. The postionization dynamics are dictated by the potential energy landscape. The D1 surface is steep downhill along the cis/trans isomerization coordinate and toward a common minimum shared by the two isomers in the region of D1/D0 conical intersection. Coherent cis/trans torsional motion along this coordinate is manifested in the ion transients by a cosine modulation. In this scenario, D0 becomes populated with molecules that are energized mainly along the cis-trans isomerization coordinate, with the kinetic energy above the cis-trans interconversion barrier. These activated azobenzene molecules easily cycle back and forth along the D0 surface and give rise to several periods of modulated signal before coherence is lost. This persistent localization of the internal energy during internal conversion is provided by the steep downhill potential energy surface, small initial internal energy content, and a strong hole-lone pair interaction that drives the molecule along the cis-trans isomerization coordinate to facilitate the transition between the involved electronic states.

Assessment of Reproducibility of Laser Electrospray Mass Spectrometry using Electrospray Deposition of Analyte.

A nonresonant, femtosecond (fs) laser is employed to desorb samples of Victoria blue deposited on stainless steel or indium tin oxide (ITO) slides using either electrospray deposition (ESD) or dried droplet deposition. The use of ESD resulted in uniform films of Victoria blue whereas the dried droplet method resulted in the formation of a ring pattern of the dye. Laser electrospray mass spectrometry (LEMS) measurements of the ESD-prepared films on either substrate were similar and revealed lower average relative standard deviations for measurements within-film (20.9%) and between-films (8.7%) in comparison to dried droplet (75.5% and 40.2%, respectively). The mass spectral response for ESD samples on both substrates was linear (R2 > 0.99), enabling quantitative measurements over the selected range of 7.0 × 10-11 to 2.8 × 10-9 mol, as opposed to the dried droplet samples where quantitation was not possible (R2 = 0.56). The limit of detection was measured to be 210 fmol. Graphical Abstract ᅟ.

Reactive Pendant Mn═O in a Synthetic Structural Model of a Proposed S4 State in the Photosynthetic Oxygen Evolving Complex.

The molecular mechanism of the Oxygen Evolving Center of photosystem II has been under debate for decades. One frequently cited proposal is the nucleophilic attack by water hydroxide on a pendant Mn═O moiety, though no chemical example of this reactivity at a manganese cubane cluster has been reported. We describe here the preparation, characterization, and a reactivity study of a synthetic manganese cubane cluster with a pendant manganese-oxo moiety. Reaction of this cluster with alkenes results in oxygen and hydrogen atom transfer reactions to form alcohol- and ketone-based oxygen-containing products. Nitrene transfer from core imides is negligible. The inorganic product is a cluster identical to the precursor, but with the pendant Mn═O moiety replaced by a hydrogen abstracted from the organic substrate, and is isolated in quantitative yield. 18O and 2H isotopic labeling studies confirm the transfer of atoms between the cluster and the organic substrate. The results suggest that the core cubane structure of this model compound remains intact, and that the pendant Mn═O moiety is preferentially reactive.

Isolating Protein Charge State Reduction in Electrospray Droplets Using Femtosecond Laser Vaporization.

Charge state distributions are measured using mass spectrometry for both native and denatured cytochrome c and myoglobin after laser vaporization from the solution state into an electrospray (ES) plume consisting of a series of solution additives differing in gas-phase basicity. The charge distribution depends on both the pH of the protein solution prior to laser vaporization and the gas-phase basicity of the solution additive employed in the ES solvent. Cytochrome c (myoglobin) prepared in solutions with pH of 7.0, 2.6, and 2.3 resulted in the average charge state distribution (Zavg) of 7.0 ± 0.1 (8.2 ± 0.1), 9.7 ± 0.2 (14.5 ± 0.3), and 11.6 ± 0.3 (16.4 ± 0.1), respectively, in ammonium formate ES solvent. The charge distribution shifted from higher charge states to lower charge states when the ES solvent contained amines additives with higher gas-phase basicity. In the case of triethyl ammonium formate, Zavg of cytochrome c (myoglobin) prepared in solutions with pH of 7.0, 2.6, and 2.3 decreased to 4.9 (5.7), 7.4 ± 0.2 (9.6 ± 0.3), and 7.9 ± 0.3 (9.8 ± 0.2), respectively. The detection of a charge state distribution corresponding to folded protein after laser vaporized, acid-denatured protein interacts with the ES solvent containing ammonium formate, ammonium acetate, triethyl ammonium formate, and triethyl ammonium acetate suggests that at least a part of protein population folds within the electrospray droplet on a millisecond timescale. Graphical Abstract ᅟ.

Gold Nanotriangle Formation through Strong-Field Laser Processing of Aqueous KAuCl4 and Postirradiation Reduction by Hydrogen Peroxide.

Femtosecond laser irradiation of aqueous KAuCl4 followed by postirradiation reduction with hydrogen peroxide (H2O2) is investigated as a new approach for the synthesis of gold nanotriangles (AuNTs) without any added surfactant molecules. Laser irradiation was applied for times ranging from 5 to 240 s, and postirradiation reduction of the solutions was monitored by UV-vis spectroscopy. Laser processing of aqueous KAuCl4 for 240 s, where the full reduction of Au(III) occurred during irradiation, produced spherical gold nanoparticles (AuNPs) with an average size of 11.4 ± 3.4 nm. Irradiation for shorter times (i.e., 15 s) resulted in the formation of laser-generated AuNP seeds (5.7 ± 1.8 nm) in equilibrium with unreacted KAuCl4 after termination of laser irradiation. The postirradiation reduction of these solutions by H2O2 produced a mixture of spherical and triangular AuNPs. Decreasing the laser irradiation time from 45 to 5 s significantly reduced the number of laser-generated Au seeds, the amount of H2O2 produced, and the rate of postirradiation reduction, resulting in the formation of a large number of AuNTs with sizes increasing from 29.5 ± 10.2 to 125 ± 43.2 nm. Postirradiation reduction is kinetically inhibited in the absence of laser-generated AuNP seeds.

Classification of Gunshot Residue Using Laser Electrospray Mass Spectrometry and Offline Multivariate Statistical Analysis.

Nonresonant laser vaporization combined with high-resolution electrospray time-of-flight mass spectrometry enables analysis of a casing after discharge of a firearm revealing organic signature molecules including methyl centralite (MC), diphenylamine (DPA), N-nitrosodiphenylamine (N-NO-DPA), 4-nitrodiphenylamine (4-NDPA), a DPA adduct, and multiple unidentified features not observed in previous mass spectral measurements. Collision-induced dissociation measurements of unknown GSR signature ions reveals inorganic barium and derivatives BaOH, BaOHCH3, BaCH3COO remaining from the primer. Both hydrophilic and hydrophobic signatures are detected using water-methanol electrospray solution. Offline principal component analysis and discrimination of the laser electrospray mass spectral (LEMS) measurements resulted in perfect classification of the gun shot residue with respect to the manufacturer. Principal component analysis of recycled and reloaded casings resulted in classification of the penultimate manufacturer with an accuracy of 89%.

Elucidating Strong Field Photochemical Reduction Mechanisms of Aqueous [AuCl4](-): Kinetics of Multiphoton Photolysis and Radical-Mediated Reduction.

Direct, multiphoton photolysis of aqueous metal complexes is found to play an important role in the formation of nanoparticles in solution by ultrafast laser irradiation. In situ absorption spectroscopy of aqueous [AuCl4](-) reveals two mechanisms of Au(0) nucleation: (1) direct multiphoton photolysis of [AuCl4](-) and (2) radical-mediated reduction of [AuCl4](-) upon multiphoton photolysis of water. Measurement of the reaction kinetics as a function of solution pH reveals zeroth-, first-, and second-order components. The radical-mediated process is found to be zeroth-order in [AuCl4](-) under acidic conditions, where the reaction rate is limited by the production of reactive radical species from water during each laser shot. Multiphoton photolysis is found to be first order in [AuCl4](-) at all pHs, whereas the autocatalytic reaction with H2O2, the photolytic reaction product of water, is second order.

Nonresonant, femtosecond laser vaporization and electrospray post-ionization mass spectrometry as a tool for biological tissue imaging.

An ambient mass spectrometry imaging (MSI) source is demonstrated with both high spatial and mass resolution that enables measurement of the compositional heterogeneity within a biological tissue sample. The source is based on nonresonant, femtosecond laser electrospray mass spectrometry (LEMS) coupled to a quadrupole time-of-flight (QTOF) mass analyzer. No matrix deposition and minimal sample preparation is necessary for the source. The laser, translation stage, and mass spectrometer are synchronized and controlled using a customized user interface. Single or multiple laser shots may be applied to each pixel. A scanning rate of 2.0s per pixel is achieved. Measurement of a patterned ink film indicates the potential of LEMS for ambient imaging with a lateral resolution of ∼60µm. Metabolites including sugar, anthocyanins and other small metabolites were successfully mapped from plant samples without oversampling using a spot size of 60×70µm(2). Molecular identification of the detected analytes from the tissue was enabled by accurate mass measurement in conjunction with tandem mass spectrometry. Statistical analysis, non-negative matrix factorization and principle component analysis, were applied to the imaging data to extract regions with distinct and/or correlated spectral profiles.

Ambient Molecular Analysis of Biological Tissue Using Low-Energy, Femtosecond Laser Vaporization and Nanospray Postionization Mass Spectrometry.

Direct analysis of plant and animal tissue samples by laser electrospray mass spectrometry (LEMS) was investigated using low-energy, femtosecond duration laser vaporization at wavelengths of 800 and 1042 nm followed by nanospray postionization. Low-energy (<50 µJ), fiber-based 1042 nm LEMS (F-LEMS) allowed interrogation of the molecular species in fresh flower petal and leaf samples using 435 fs, 10 Hz bursts of 20 pulses from a Ytterbium-doped fiber laser and revealed comparable results to high energy (75-1120 µJ), 45 fs, 800 nm Ti:Sapphire-based LEMS (Ti:Sapphire-LEMS) measurements. Anthocyanins, sugars, and other metabolites were successfully detected and revealed the anticipated metabolite profile for the petal and leaf samples. Phospholipids, especially phosphatidylcholine, were identified from a fresh mouse brain section sample using Ti:Sapphire-LEMS without the application of matrix. These lipid features were suppressed in both the fiber-based and Ti:Sapphire-based LEMS measurements when the brain sample was prepared using the optimal cutting temperature compounds that are commonly used in animal tissue cryosections.

Filament-Assisted Impulsive Raman Spectroscopy of Ozone and Nitrogen Oxides.

The filament-assisted impulsive Raman spectra of ozone, nitric oxide, and nitrogen dioxide are presented. The Raman response as a function of ozone concentration scales as N(2), where N is the number of oscillators in the interaction region. The system described has a detection limit of ∼300 ppm for gas-phase ozone. Ozone produced via the strong field chemistry occurring within the filament pump was also detected. The measurements reveal spectral interference in the Raman features. Simulations show the spectral fringing results from interference of the Raman signal with pump-induced cross-phase modulation. The fringes are used to classify the symmetric mode of the low concentration filament-generated ozone.

High-energy noncollinear optical parametric amplifier producing 4 fs pulses in the visible seeded by a gas-phase filament.

We report on the design and characterization of a short-pulse-pumped, single-stage noncollinear optical parametric amplifier (NOPA) that achieves high pulse energies in the few-cycle pulse regime. Optimal pulse-front tilting and temporal compression of the short (35 fs) pump pulse are achieved using a 4f grating compressor, while spatial chirp at the NOPA crystal is eliminated with proper imaging using a pair of reflective telescopes. Gas-phase filamentation in an open-ended argon-filled cell provides a bright, stable seed source with little residual chirp that is suitable for temporal overlap with the short pump pulse without dispersion precompensation. Two seeding geometries are explored, and pulses as short as 3.5 fs are obtained by seeding with the entire filament bandwidth. Fourier-transform-limited 4 fs pulses are obtained by filtering the IR portion of the spectrum.

Triangular gold nanoplate growth by oriented attachment of Au seeds generated by strong field laser reduction.

The synthesis of surfactant-free Au nanoplates is desirable for the development of biocompatible therapeutics/diagnostics. Rapid Δ-function energy deposition by irradiation of aqueous KAuCl4 solution with a 5 s burst of intense shaped laser pulses, followed by slow addition of H2O2, results in selective formation of nanoplates with no additional reagents. The primary mechanism of nanoplate formation is found to be oriented attachment of the spherical seeds, which self-recrystallize to form crystalline Au nanoplates.

Increasing protein charge state when using laser electrospray mass spectrometry.

Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol (m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

Internal energy deposition for low energy, femtosecond laser vaporization and nanospray post-ionization mass spectrometry using thermometer ions.

The internal energy of p-substituted benzylpyridinium ions after laser vaporization using low energy, femtosecond duration laser pulses of wavelengths 800 and 1042 nm was determined using the survival yield method. Laser vaporization of dried benzylpyridinium ions from metal slides into a buffered nanospray with 75 µJ, 800 nm laser pulses resulted in a higher extent of fragmentation than conventional nanospray due to the presence of a two-photon resonance fragmentation pathway. Using higher energy 800 nm laser pulses (280 and 505 µJ) led to decreased survival yields for the four different dried benzylpyridinium ions. Analyzing dried thermometer ions with 46.5 µJ, 1042 nm pulse-bursts resulted in little fragmentation and mean internal energy distributions equivalent to nanospray, which is attributable to the absence of a two-photon resonance that occurs with higher energy, 800 nm laser pulses. Vaporization of thermometer ions from solution with either 800 nm or 1042 nm laser pulses resulted in comparable internal energy distributions to nanospray ionization.