Distributions Of Fragment Ions Research Articles

Advancing Protein Analysis: A Low-Pressure Drift Tube Orbitrap Mass Spectrometer for Ultraviolet Photodissociation-Based Structural Characterization.

Owing to its ability to generate extensive fragmentation of proteins, ultraviolet photodissociation (UVPD) mass spectrometry (MS) has emerged as a versatile ion activation technique for the structural characterization of native proteins and protein complexes. Interpreting these fragmentation patterns provides insight into the secondary and tertiary structures of protein ions. However, the inherent complexity and diversity of proteins often pose challenges in resolving their numerous conformations. To address this limitation, we combined UVPD-MS with drift tube ion mobility, offering potential to acquire conformationally selective MS/MS information. A low-pressure drift tube (LPDT) Orbitrap mass spectrometer equipped with 193 nm UVPD capabilities enables the analysis of protein conformers through the analysis of arrival time distributions (ATDs) of individual fragment ions. ATDs of fragment ions are compared for different backbone cleavage sites of the protein or different precursor charge states to give information about regions of potential folding or elongation. This integrated platform offers promise for advancing our understanding of protein structures in the gas phase.

Size limits and fission channels of doubly charged noble gas clusters.

Small, highly charged liquid droplets are unstable with respect to spontaneous charge separation when their size drops below the Rayleigh limit or, in other words, their fissility parameter X exceeds the value 1. The absence of small doubly charged atomic cluster ions in mass spectra below an element-specific appearance size na has sometimes been attributed to the onset of barrierless fission at X = 1. However, more realistic models suggest that na marks the size below which the rate of fission surpasses that of competing dissociative channels, and the Rayleigh limit of doubly charged van der Waals clusters has remained unchartered. Here we explore a novel approach to form small dicationic clusters, namely by Penning ionization of singly charged noble gas (Ng) clusters that are embedded in helium nanodroplets; the dications are then gently extracted from the nanodroplets by low-energy collisions with helium gas. We observe Ngn2+ ions that are about 40% smaller than previously reported for xenon and krypton and about 20% for argon. These findings suggest that fission barriers have been underestimated in previous theoretical work. Furthermore, we measure the size distributions of fragment ions that are produced by collisional excitation of mass-selected dications. At lowest collision gas pressure, dicationic Kr and Xe clusters that are smaller than previously observed are found to evaporate an atom before they undergo highly symmetric fission. The distribution of fragments resulting from fission of small dicationic Ar clusters is bimodal.

Strong-field photoionization/dissociation of vinyl bromide in near-infrared and ultraviolet femtosecond laser fields

Ionization of polyatomic molecules in strong femtosecond laser fields is a pivotal process that may induce a variety of highly nonlinear molecular physical phenomena. We performed an experimental study on photoionization and dissociation of vinyl bromide molecules in 800 nm or 400 nm femtosecond strong laser fields using a time-of-flight mass spectrometer. The dependence of the ion yields on laser intensity and polarization was investigated, and the results are compared in different wavelength laser fields. The kinetic energy releases from Coulomb explosion of vinyl bromide, and the angular distributions of different fragment ions were obtained. The mechanism of strong-field ionization/dissociation of vinyl bromide in different laser fields is discussed based on the experimental results.

Ultraviolet Photodissociation Reveals the Molecular Mechanism of Crown Ether Microsolvation Effect on the Gas-Phase Native-like Protein Structure.

Maintaining the protein high-order structures and interactions during the transition from aqueous solution to gas phase is essential to the structural analysis of native mass spectrometry (nMS). Herein, we systematically interrogate the effects of charge state and crown ether (CE) complexation on the gas-phase native-like protein structure by integrating nMS with 193 nm ultraviolet photodissociation (UVPD). The alterations of photofragmentation yields of protein residues and the charge site distribution of fragment ions reveal the specific sites and sequence regions where charge and CE take effect. Our results exhibit the CE complexation on protonated residues can largely alleviate the structure disruption induced by the intramolecular solvation of charged side chains. The influences of CE complexation and positive charge on gas-phase protein structure exhibit generally opposite trends because the CE microsolvation avoids the hydrogen-bonding formation between the charged side chains with backbone carbonyls. Thus, CE complexation leads to a more stable and native-like protein structure in the gas phase.

Conditional Fragment Ion Probabilities Improve Database Searching for Nonmonoisotopic Precursors.

Stochastic, intensity-based precursor isolation can result in isotopically enriched fragment ions. This problem is exacerbated for large peptides and stable isotope labeling experiments using deuterium or 15N. For stable isotope labeling experiments, incomplete and ubiquitous labeling strategies result in the isolation of peptide ions composed of many distinct structural isomers. Unfortunately, existing proteomics search algorithms do not account for this variability in isotopic incorporation, and thus often yield poor peptide and protein identification rates. We sought to resolve this shortcoming by deriving the expected isotopic distributions of each fragment ion and incorporating them into the theoretical mass spectra used for peptide-spectrum-matching. We adapted the Comet search platform to integrate a modified spectral prediction algorithm we term Conditional fragment Ion Distribution Search (CIDS). Comet-CIDS uses a traditional database searching strategy, but for each candidate peptide we compute the isotopic distribution of each fragment to better match the observed m/z distributions. Evaluating previously generated D2O and 15N labeled data sets, we found that Comet-CIDS identified more confident peptide spectral matches and higher protein sequence coverage compared to traditional theoretical spectra generation, with the magnitude of improvement largely determined by the amount of labeling in the sample.

Multichannel dissociative ionization of ethanol in intense ultraviolet laser fields: Energy correlation between photoelectron emission and fragment recoil

We investigate multichannel dissociative ionization of ethanol in intense ultraviolet laser fields using a photoelectron-photoion coincidence momentum imaging technique. Product channels are clearly separated with detected photoions, and channel-specific photoelectron spectra exhibit multiple spectral components unambiguously assigned to four- and five-photon ionization to the electronic ground state and the electronically excited state. We measure kinetic energy distributions of fragment ions as a function of the energy of a correlated photoelectron to reveal how much energy is provided to the fragment recoil of fragment ions in the course of photoelectron emission and subsequent electronic excitation. Subsequent electronic excitation rather than photoelectron emission governs the internal energy of ethanol cations. The role of subsequent electronic excitation becomes more decisive as the laser intensity increases.

Fast screening and confirmation of 73 common prohibited compounds in cosmetics by ultra performance liquid chromatography-quadrupole-time-of-flight high resolution mass spectrometry

建立了超高效液相色谱-四极杆-飞行时间高分辨质谱(UPLC-Q-TOF HRMS)同时快速筛查确证化妆品中73种常见禁用物质的方法。样品经饱和氯化钠溶液分散均匀后,采用含0.2%甲酸的乙腈溶液超声提取,50 mg PSA净化,以8000 r/min高速冷冻离心除脂,采用Waters Acquity HSS T3色谱柱(100 mm×2.1 mm, 1.8 μm)分离。采用多反应监测高分辨扫描模式(MRM HR),以保留时间、一级母离子精确质量数、同位素丰度比和二级子离子精确质量数实现化妆品中73种禁用物质的快速筛查和确证,基质匹配外标法定量。实验比较了不同提取溶剂、净化吸附剂、色谱条件和质谱扫描模式对73种禁用物质测定的影响,并考察了膏霜剂和水剂的基质效应。结果表明,73种禁用物质线性关系良好,相关系数(R2)>0.99;检出限为5~150 μg/kg;定量限为15~450 μg/kg;膏霜剂及水剂两种基质在3个加标水平下的回收率为60.3%~130.3%,日内、日间RSD分别为0.8%~10.0%(n=6)和1.1%~15.0%(n=3)。日常风险监测中检出磺胺甲基异噁唑、甲基泼尼松、林可霉素、对乙酰氨基酚、甲氧苄啶、阿法骨化醇、倍他米松戊酸酯、溴莫尼定、氯霉素、氯苯那敏、氯倍他索丙酸酯、克罗米通、益康唑、酮康唑、泼尼松醋酸酯和泼尼松,检出含量范围为0.5~1136.1 mg/kg。该方法准确、快速、简便,可用于化妆品中73种常见禁用物质的检测。

Ro-vibrational Distribution of NO+ Dissociated from NO2+ Ions in the a3B2 and b3A2 States: A Slow "Impulsive" Dissociation Example Revealed from Threshold Photoelectron-Photoion Coincidence Imaging.

To clarify the contentions about dissociative photoionization mechanism of nitrogen dioxide via the a3B2 and b3A2 ionic states, a new threshold photoelectron-photoion coincidence (TPEPICO) velocity imaging has been conducted in the 12.8-14.0 eV energy range at the Hefei Light Source. The fine vibrational-resolved threshold photoelectron spectrum agrees well with the previous measurements. The ro-vibrational distributions of NO+, as the unique fragment ion in the dissociation of NO2+ in specific vibronic levels of a3B2 and b3A2 states, are derived from the recorded TPEPICO velocity images. A "cold" vibrational (v+ = 0) and "hot" rotational population is observed at the a3B2(0,3,0) and (0,4,0) vibronic levels, while the dissociation of NO2+ in b3A2(0,0,0) leads to the NO+ fragment with both hot vibrational and rotational populations. With the aid of the quantum chemical calculations at the time-dependent B3LYP level, minimum energy paths on the potential energy surfaces of the a3B2 and b3A2 states clarify their adiabatic dissociation mechanisms near the thresholds, and this study proposes reliable explanations for the observed internal energy distributions of fragment ions. Additionally, this study provides valuable insights into the application of the classical "impulsive" model on an overall slow dissociation process.

Dissociative Ionization of Molecular CF2Br2 under 800 and 400 nm Intense Femtosecond Laser Fields

The interaction between the CF2Br2 molecule and 800/400 nm intense femtosecond laser fields is investigated by direct current (dc) sliced velocity mapping imaging implementation. By analyzing the kinetic energy release distribution and angular distribution of fragment ions, the dissociation channels along C-Br bond cleavage have been determined. The isotropic structure of the angular distribution for CF2Br+ ions is attributed to the coupling between the excited states. Additionally, a unique elimination channel of CF2Br2+ → CF2 + Br2+ has been observed and identified in the case of 400 nm laser field, in which the two C-Br bonds break asynchronously.

Screening and quantitative analysis of nine illicit antiallergics in emulsion cosmetics by ultra-high performance liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry

A rapid and accurate analysis method based on ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight high-resolution mass spectrometry (UPLC-Q-TOF-HRMS) was developed to screen and determine nine antiallergy drugs in emulsion cosmetics. First, a standard library of the target compounds was established. The library contained the TOF-MS information and secondary MS information such as retention time, ion addition mode, mass error, isotope distribution, mass-to-charge ratio of the parent ion, and fragment ion distribution. According to the European Union regulation (SANTE/11945/2015), the standard for the qualitative determination by HRMS was determined; that is, each compound was confirmed by two ions with a mass error below 5%, and the abundance ratio of the two ions was less than 30%. Second, the instrument conditions and sample pretreatment conditions for the determination of different compounds were optimized, and the influence of different levels of quantitative ions on the matrix effect was compared. The following observations were made:(1) the addition of 0.1% formic acid to the water phase improved the response of the chromatographic peaks; (2) among the various solvent amounts tested (4, 5, 6, 8 mL acetonitrile and 4, 5, 6, 8 mL methanol), 4 mL acetonitrile showed the best extraction efficiency; (3) PRiME HLB had a better purification effect than the other two purification columns (C18 and HLB solid-phase extraction cartridges), thus reducing the interference of impurities and ensuring good recovery of the target compounds; (4) the use of two pairs of secondary product ion quantification could significantly reduce the matrix effect of anti-allergic compounds and improve the quantification accuracy. Finally, based on the above findings, the experimental procedure was established. The lotion samples were first ultrasonically extracted with acetonitrile and purified on the PRiME HLB column. Chromatographic separation was performed on a Waters XBridge C18 column with gradient elution using 0.1% (v/v) formic acid in water and acetonitrile. Finally, the sequential window acquisition of all theoretical mass spectra (SWATH), which shows obvious advantages in continuous and high-throughput acquisition, was selected for MS data acquisition. The retention time, mass accuracy, isotope distribution, and fragment ion matching ratio were used for fast qualitative screening, while the peak areas of characteristic product ions were used for precise quantification. All the calibration curves showed good linearity (r2>0.99) within the tested ranges (5-100 μg/L) under the optimum conditions. The limits of quantification (LOQs) were in the range of 0.05-0.10 mg/kg. The recoveries were in the range of 65.3%-107% at three spiked levels (0.10, 0.20, and 0.60 mg/kg), with relative standard deviations (RSDs, n=6) below 20%. Compared with the existing ion exchange column methods, the proposed "one-step" purification method based on PRiME HLB is simpler and more rapid, where the extraction solution is filtered directly after allowing it to pass through the column, without any subsequent washing and elution procedures. In addition, the LOQs of this method are lower than those of other LC-MS/MS methods, indicating that the proposed method has higher sensitivity. The application of SWATH data acquisition makes it possible to achieve quantification with two pairs of product ions, thus reducing the matrix effect and ensuring accuracy of the quantitative results. Therefore, the proposed method is less time-consuming and operationally convenient, and it can be used for the rapid screening and accurate quantification of antiallergics in lotion samples.

Analysis of kerogens and model compounds by time-of-flight secondary ion mass spectrometry (TOF-SIMS)

Here, kerogens of differing heat treatments are subjected to extremely high dissociation energies by sample bombardment by 25 keV Bi3+ primary ions during analysis by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Positive and negative secondary ions are produced from this decomposition and fragment ion distributions of model compounds and kerogens are compared and starkly different results are obtained for cations versus anions. Cations exhibit a large range of C/H ratios and include highly unsaturated linear chain ions and aromatic ions. Cations of kerogens possess predominantly no heteroatoms. Positive fragment ion distributions depend on the source material being bombarded. Mature, more aromatic kerogens produce higher yields of fragment ions of highly unsaturated carbon chains while immature, more aliphatic kerogens produce more aromatic fragment ions, particularly at the higher carbon numbers. This is consistent with the observation that aromatic model compounds produce a greater fraction of hydrogen-deficient, carbon chain fragment ions, as compared to a purely aliphatic model compound. There is substantial suppression of free radical fragment cations, except for large fragments. In contrast, there is little free radical suppression of the anions. The anions tend to be very hydrogen deficient, spanning a small range of C/H ratios. Highly unsaturated to pure carbon chain fragment anions dominate while aromatic anions are not found. In both positive and negative ion spectra, the yields of fragment ions corresponding to derivatives of the carbon chain molecules, polyynes and allenes, are substantial. Some heteroatom-containing fragment anions are produced, all of which are very hydrogen deficient.

Ionic Angular Distributions Induced by Strong-Field Ionization of Tri-Atomic Molecules* *Supported by the National Natural Science Foundation of China (Grant Nos. 11534004, 11874179, and 11704149), and the Natural Science Foundation of Jilin Province (Grant Nos. 20180101289JC and 20190103045JH).

Angular distributions of fragment ions from ionization of several tri-atomic molecules (CO 2, OCS, N 2 O and NO 2 ) by strong 800-nm laser fields are investigated via a time-of-flight mass spectrometer. Anisotropic angular distributions of fragment ions, especially those of atomic ions, are observed for all of the molecules studied. These anisotropic angular distributions are mainly due to the geometric alignment of molecules in the strong field ionization. Distinct different patterns in ionic angular distributions for different molecules are observed. It is indicated that both molecular geometric structure and ionization channels have effects on the angular distributions of strong field ionization/fragmentation.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z′ ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH3 fragment ions (“twins”). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH3 ions requires consideration for accurate and confident assignments of z′ ions in MALDI-ISD FT-ICR mass spectra.

Imaging drugs, metabolites and biomarkers in rodent lung: a DESI MS strategy for the evaluation of drug-induced lipidosis

Within drug development and pre-clinical trials, a common, significant and poorly understood event is the development of drug-induced lipidosis in tissues and cells. In this manuscript, we describe a mass spectrometry imaging strategy, involving repeated analysis of tissue sections by DESI MS, in positive and negative polarities, using MS and MS/MS modes. We present results of the detected distributions of the administered drug, drug metabolites, lipid molecules and a putative marker of lipidosis, di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate (di-22:6-BMP). A range of strategies have previously been reported for detection, isolation and identification of this compound, which is an isomer of di-docosahexaenoic (22:6 n-3) phosphatidylglycerol (di-22:6 PG), a commonly found lipid that acts as a surfactant in lung tissues. We show that MS imaging using MS/MS can be used to differentiate these compounds of identical mass, based upon the different distributions of abundant fragment ions. Registration of images of these fragments, and detected drugs and metabolites, is presented as a new method for studying drug-induced lipidosis in tissues.Graphical abstract

Elucidating Branching Topology and Branch Lengths in Star-Branched Polymers by Tandem Mass Spectrometry.

Tandem mass spectrometry (MS2) has been employed to elucidate the topology and branching architecture of star-branched polyethers. The polymers were ionized by matrix-assisted laser desorption/ionization (MALDI) to positive ions and dissociated after leaving the ion source via laser-induced fragmentation. The bond scissions caused under MALDI-MS2 conditions occur preferentially near the core-branch joining points due to energetically favorable homolytic and heterolytic bond cleavages near the core and release of steric strain and/or reduction of crowding. This unique fragmentation mode detaches complete arms from the core generating fragment ion series at the expected molecular weight of each branch. The number of fragment ion distributions observed combined with their mass-to-charge ratios permit conclusive determination of the degree of branching and the corresponding branch lengths, as demonstrated for differently branched homo- and mikto-arm polyether stars synthesized via azide-alkyne click chemistry. The results of this study underscore the utility of MS2 for the characterization of branching architecture and branch lengths of (co) polymers with two or more linear chains attached to a functionalized central core.

Proteome dynamics from heavy water metabolic labeling and peptide tandem mass spectrometry

Protein homeostasis (proteostasis) is a result of a dynamic equilibrium between protein synthesis and degradation. It is important for healthy cell/organ functioning and is often associated with diseases such as neurodegenerative diseases and non-Alcoholic Fatty Liver disease. Heavy water metabolic labeling, combined with liquid-chromatography and mass spectrometry (LC-MS), is a powerful approach to study proteostasis in vivo in high throughput. Traditionally, intact peptide signals are used to estimate stable isotope incorporation in time-course experiments. The time-course of label incorporation is used to extract protein decay rate constant (DRC). Intact peptide signals, computed from integration in chromatographic time and mass-to-charge ratio (m/z) domains, usually, provide an accurate estimate of label incorporation. However, sample complexity (co-elution), limited dynamic range, and low signal-to-noise ratio (S/N) may adversely interfere with the peptide signals. These artifacts complicate the DRC estimations by distorting peak shape in chromatographic time and m/z domains. Fragment ions, on the other hand, are less prone to these artifacts and are potentially well suited in aiding DRC estimations. Here, we show that the label incorporation encoded into the isotope distributions of fragment ions reflect the isotope enrichment during the metabolic labeling with heavy water. We explore the label incorporation statistics for devising practical approaches for DRC estimations.

Hydrogen-Deuterium Exchange and Electron Capture Dissociation to Interrogate the Conformation of Gaseous Melittin Ions.

There is a need in the field of biological mass spectrometry for structural tools which can report on regional, rather than solely global, structure of gaseous protein ions. Site-specific hydrogen-deuterium (H/D) exchange has shown promise in fulfilling this need, but requires additional method development to prove its utility. In this study, we use H/D exchange and electron capture dissociation (ECD) to probe the gaseous structure of two peptides which are α-helical in solution and which differ by a single point mutation. Global H/D exchange levels, ECD fragmentation profiles, and region specific H/D exchange profiles are compared between wild type (WT) melittin, which adopts a hinged helix conformation in solution, and a mutant P14A melittin which folds into a single helix in solution. High protection from H/D exchange by both peptides is consistent with retention of secondary structure in the gas phase (or refolding into some other compact structure). The P14A mutant melittin exhibits lower ECD fragmentation efficiency than WT melittin, suggesting that it contains more secondary structure in the gas phase, which may indicate that these peptides retain some memory of their solution-phase structures. Examination of the isotopic distributions of fragment ions derived from H/D exchange with subsequent ECD reveals that the C-terminus of these peptides adopts multiple conformations. The results reported here offer insight into the stability of alpha helices in the gas phase, and also highlight the value of combining gas-phase H/D exchange with electron capture dissociation to interrogate gaseous peptide conformation.

Top-Down Analysis of Proteins in Low Charge States.

The impact of charging methods on the dissociation behavior of intact proteins in low charge states is investigated using HCD and 193nm UVPD. Low charge states are produced for seven different proteins using the following four different methods: (1) proton transfer reactions of ions in high charge states generated from conventional denaturing solutions; (2) ESI of proteins in solutions of high ionic strength to enhance retention of folded native-like conformations; (3) ESI of proteins in high pH solutions to limit protonation; and (4) ESI of carbamylated proteins. Comparison of sequence coverages, degree of preferential cleavages, and types and distribution of fragment ions reveals a number of differences in the fragmentation patterns depending on the method used to generate the ions. More notable differences in these metrics are observed upon HCD than upon UVPD. The fragmentation caused by HCD is influenced more significantly by the presence/absence of mobile protons, a factor that modulates the degree of preferential cleavages and net sequence coverages. Carbamylation of the lysines and the N-terminus of the proteins alters the proton mobility by reducing the number of proton-sequestering, highly basic sites as evidenced by decreased preferential fragmentation C-terminal to Asp or N-terminal to Pro upon HCD. UVPD is less dependent on the method used to generate the low charge states and favors non-specific fragmentation, an outcome which is important for obtaining high sequence coverage of intact proteins.

Dissociation dynamics of energy-selected ions using threshold photoelectron-photoion coincidence velocity imaging

Threshold photoelectron-photoion coincidence (TPEPICO) is a powerful method to prepare and analyze internal energy- or state-selected ions. Here, we review the state-of-the-art TPEPICO imaging technique combining with tunable vacuum ultraviolet (VUV) synchrotron radiation and its recent applications at Hefei Light Source (HLS), especially on the fundamental data measurement and the dissociation dynamics of ions. By applying the double velocity map imaging for both electrons and ions in coincidence, the collection efficiency of the charged particles, the electron energy resolution and the resolving power of the released kinetic energy in dissociation have been greatly improved. The kinetic energy and the angular distributions of fragment ions dissociated from parent ions with definitive internal energy or state have been acquired directly from TPEPICO images. Some dissociation mechanisms involving non-adiabatic quantum effects, like conical intersection and internal conversion, have been revealed. Moreover, the mass-selected threshold photoelectron spectroscopy (MS-TPES) shows tremendous advantages in isomer-specific analysis of complex systems.

Multiorbital effects in strong-field ionization and dissociation of aligned polar molecules CH3I and CH3Br

Controlling the molecular axis offers additional ways to study molecular ionization and dissociation in strong laser fields. We measure the ionization and dissociation yields of aligned polar ${\mathrm{CH}}_{3}X$ ($X=\mathrm{I}$, Br) molecules in a linearly polarized femtosecond laser field. The current data show that maximum ionization occurs when the laser polarization is perpendicular to the molecular $\mathrm{C}\ensuremath{-}X$ axis, and dissociation prefers to occur at the laser polarization parallel to the $\mathrm{C}\ensuremath{-}X$ axis. The observed angular distributions suggest that the parent ions are generated by ionization from the HOMO. The angular distribution of fragment ions indicates that dissociation occurs mainly from an ionic excited state produced by ionization from the HOMO-1.