Collision-induced Dissociation Measurements Research Articles

Defining the Ceramide Composition of Bovine and Human Milk Gangliosides by Direct Infusion ESI-CID Tandem Mass Spectrometry of Native and Permethylated Molecular Species.

Gangliosides are glycosphingolipids composed of an oligosaccharide that contains one or more sialic acid residues and is linked to a ceramide, a lipid composed of a long chain base (LCB) that bears an amide-linked fatty acyl group (FA). The ceramide portions of gangliosides are embedded in cell membranes; the exposed glycans interact with the extracellular environment. Gangliosides play a myriad of roles in activities such as cell-cell communication, formation of lipid rafts, cellular adhesion, calcium homeostasis, host-pathogen interaction, and viral invasion. Although the epitopes responsible for the interactions of gangliosides are located in the glycan, the epitope presentation is strongly influenced by the orientation of the attached ceramide within the lipid membrane, a feature that depends on the details of its structure, that is, the specific LCB and FA. Since the identities of both the glycan and the ceramide affect the activity of gangliosides, it is important to characterize the individual intact molecular forms. We report here a mass spectrometry-based method that combines the information gained from low-energy collision-induced dissociation (CID) measurements for the determination of the glycan with tandem mass spectra obtained at stepped higher-energy CID for the detailed characterization of the LCB and FA components of intact gangliosides. We provide results from applications of this method to the analysis of gangliosides present in bovine and human milk in order to demonstrate the assignment of LCB and FA for intact gangliosides and differential detection of isomeric ceramide structures.

Photodissociation Spectroscopy and Photofragment Imaging to Probe Fe+(Benzene)1,2 Dissociation Energies.

Tunable laser photodissociation spectroscopy measurements and photofragment imaging experiments are employed to investigate the dissociation energy of the Fe+(benzene) ion-molecule complex. Additional spectroscopy measurements determine the dissociation energy of Fe+(benzene)2. The dissociation energies for Fe+(benzene) determined from the threshold for the appearance of the Fe+ fragment (48.4 ± 0.2 kcal/mol) and photofragment imaging (≤49.3 ± 3.2 kcal/mol) agree nicely with each other and with the value determined previously by collision-induced dissociation (49.5 ± 2.9 kcal/mol), but they are lower than the values produced by computational chemistry at the density functional theory level using different functionals recommended for transition-metal chemistry. The threshold measurement for Fe+(benzene)2 (43.0 ± 0.2 kcal/mol) likewise agrees with the value (44.7 ± 3.8 kcal/mol) from previous collision-induced dissociation measurements.

Protonated Forms of Naringenin and Naringenin Chalcone: Proteiform Bioactive Species Elucidated by IRMPD Spectroscopy, IMS, CID-MS, and Computational Approaches.

Naringenin (Nar) and its structural isomer, naringenin chalcone (ChNar), are two natural phytophenols with beneficial health effects belonging to the flavonoids family. A direct discrimination and structural characterization of the protonated forms of Nar and ChNar, delivered into the gas phase by electrospray ionization (ESI), was performed by mass spectrometry-based methods. In this study, we exploit a combination of electrospray ionization coupled to (high-resolution) mass spectrometry (HR-MS), collision-induced dissociation (CID) measurements, IR multiple-photon dissociation (IRMPD) action spectroscopy, density functional theory (DFT) calculations, and ion mobility-mass spectrometry (IMS). While IMS and variable collision-energy CID experiments hardly differentiate the two isomers, IRMPD spectroscopy appears to be an efficient method to distinguish naringenin from its related chalcone. In particular, the spectral range between 1400 and 1700 cm-1 is highly specific in discriminating between the two protonated isomers. Selected vibrational signatures in the IRMPD spectra have allowed us to identify the nature of the metabolite present in methanolic extracts of commercial tomatoes and grapefruits. Furthermore, comparisons between experimental IRMPD and calculated IR spectra have clarified the geometries adopted by the two protonated isomers, allowing a conformational analysis of the probed species.

Breath response following a nutritional challenge monitored by secondary electrospray ionization high-resolution mass spectrometry

On-line breath analysis using secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS) is a sensitive method for biomarker discovery. The strengths of this technology have already been demonstrated in the clinical environment. For the first time, this study demonstrates the application of SESI-HRMS in the field of nutritional science using a standardized nutritional intervention, consisting of a high-energy shake (950 kcal, 8% protein, 35% sugar and 57% fat). Eleven subjects underwent the intervention on three separate days and their exhaled breath was monitored up to six hours postprandially. In addition, sampling was performed during equivalent fasting conditions for selected subjects. To estimate the impact of inter- and intra-individual variability, analysis of variance simultaneous component analysis was conducted, revealing that the inter-individual variability accounted for 30% of the data variation. To distinguish the effect of the intervention from fasting conditions, partial least squares discriminant analysis was performed. Candidate compound annotation was performed with pathway analysis and collision-induced dissociation (CID) experiments. Pathway analysis highlighted, among others, features associated with the metabolism of linoleate, butanoate and amino sugars. Tentative compounds annotated through CID measurements include fatty acids, amino acids, and amino acid derivatives, some of them likely derived from nutrients by the gut microbiome (e.g. propanoate, indoles), as well as organic acids from the Krebs cycle. Time-series clustering showed an overlap of observed kinetic trends with those reported previously in blood plasma.

Structures, proton transfer and dissociation of hydroxylammonium nitrate (HAN) revealed by electrospray ionization tandem mass spectrometry and molecular dynamics simulations.

Hydroxylammonium nitrate (HAN) is a potential propellant candidate for dual-mode propulsion systems that combine chemical and electrospray thrust capabilities for spacecraft applications. However, the electrospray dynamics of HAN is currently not well understood. Capitalizing on electrospray ionization guided-ion beam tandem mass spectrometry and collision-induced dissociation measurements, and augmented by extensive molecular dynamics simulations, this work characterized the structures and reaction dynamics of the species present in the electrosprays of HAN under different conditions, which mimic those possibly occurring in low earth orbit and outer space. While being ionic in nature, the HAN monomer, however, adopts a stable covalent structure HONH2·HNO3 in the gas phase. Spontaneous proton transfer between the HONH2 and HNO3 moieties within the HAN monomer can be induced in the presence of a NO3-, a water ligand or a second HAN monomer within 3-5 Å or a H+ within 8 Å, regardless of their collision impact parameters. These facts imply that HAN proton transfer is trigged by a charge and/or a dipole of the collision partner without the need of chemical interaction or physical contact. Moreover, the addition of NO3- to HAN leads to the formation of a stable -O3N·HONH3+·NO3- anion in negative electrosprays. In contrast, when a proton approaches the HONH2·HNO3 structure, dissociative reactions occur that lead to the H2O, NO2 and HONH2 fragments (and their cations) but not intact HAN species in positive electrosprays.

Non-statistical fragmentation in photo-activated flavin mononucleotide anions.

The spectroscopy and photo-induced dissociation of flavin mononucleotide anions in vacuo are investigated over the 300-500nm wavelength range. Comparison of the dependence of fragment ion yields as a function of deposited photon energy with calculated dissociation energies and collision-induced dissociation measurements performed under single-collision conditions suggests that a substantial fraction of photo-activated ions decompose through non-statistical fragmentation pathways. Among these pathways is the dominant photo-induced fragmentation channel, the loss of a fragment identified as formylmethylflavin. The fragment ion specific action spectra reveal electronic transition energies close to those for flavins in solution and previously published gas-phase measurements, although the photo-fragment yield upon excitation of the S2 ← S0 transition appears to be suppressed.

Ligand effects in gold-carbonyl complexes: Evaluation of the bond dissociation energies using blackbody infrared radiative dissociation

In a previous work, the ability of gold-carbonyl bond dissociation energies (BDEs) to describe ligand effects in gold(I) complexes was probed using collision-induced dissociation measurements. Despite the large experimental error of the technique, results agreed with theoretical description of these effects. We propose here another experimental approach to evaluate the BDEs of [LAu-CO]+ complexes, by using Blackbody Infrared Radiative Dissociation (BIRD) method which is able to provide absolute energetic measurements. To this aim, the dissociation of 8 gold-carbonyl complexes was studied with this approach to obtain their Arrhenius activation energies. Because these complexes do not reach the rapid energy exchange (REX) limit conditions, critical energies of these dissociations were then determined using the kinetic data and an Equilibrium Truncated Thermal (ETT) internal energy distribution model implemented in the RRKM-QET MassKinetics software. To validate our approach, the known critical energies of proton-bound amino acid dimers were first reinvestigated using this model. The results are in excellent agreement with those reported in the literature. This indicates that the assumptions of this simple model are reasonable and allow the determination of accurate dissociation energies. For [LAu-CO]+ complexes, even though a shift (from 0.18 to 0.26 eV) is observed between binding energies obtained and calculated values, a linear tendency between these two sets of data is still obtained. These results validate the use of BIRD in combination with equilibrium truncated thermal internal energy distributions model to obtain dissociation energies for relatively small ions. It is also shown herein that the measure of the gold-carbonyl bond dissociation energies is a good descriptor of ligand effects in gold(I) complexes that may be reliably evaluated using the BIRD technique.

Origin and Prediction of Highly Specific Bond Cleavage Sites in the Thermal Activation of Intact Protein Ions.

Predicting the fragmentation patterns of proteins would be beneficial for the reliable identification of intact proteins by mass spectrometry. However, the ability to accurately make such predictions remains elusive. An approach to predict the specific cleavage sites in whole proteins resulting from collision-induced dissociation by use of an improved electrostatic model for calculating the proton configurations of highly-charged protein ions is reported. Using ubiquitin, cytochrome c, lysozyme and β-lactoglobulin as prototypical proteins, this approach can be used to predict the fragmentation patterns of intact proteins. For sufficiently highly charged proteins, specific cleavages occur near the first low-basicity amino acid residues that are protonated with increasing charge state. Hybrid QM/QM' (QM=quantum mechanics) and molecular dynamics (MD) simulations and energy-resolved collision-induced dissociation measurements indicated that the barrier to the specific dissociation of the protonated amide backbone bond is significantly lower than competitive charge remote fragmentation. Unlike highly charged peptides, the protons at low-basicity sites in highly charged protein ions can be confined to a limited sequence of low-basicity amino acid residues by electrostatic repulsion, which results in highly specific fragmentation near the site of protonation. This research suggests that the optimal charge states to form specific sequence ions of intact proteins in higher abundances than the use of less specific ion dissociation methods can be predicted a priori.

Electrospray mass spectrometry and molecular modeling study of formation and stability of silver complexes with diazaperylene and bisisoquinoline.

The complex formation of the following diazaperylene ligands (L) 1,12-diazaperylene 1, 1,1'-bisisoquinoline 2, 2,11-disubstituted 1,12-diazaperylenes (alkyl=methyl, ethyl, isopropyl, 3, 5, 7), 3,3'-disubstituted 1,1'-bisisoquinoline (alkyl=methyl, ethyl, isopropyl, 4, 6, 8 and with R=phenyl, 11 and with pyridine 12), and the 5,8-dimethoxy-substituted diazaperylene 9, 6,6'-dimethoxy-substituted bisisoquinoline 10 with AgBF4 was investigated. Collision-induced dissociation measurements were used to evaluate the relative stabilities of the ligands themselves and for the [1:1]+ complexes as well as for the homoleptic and heteroleptic silver [1:2]+ complexes in the gas phase. This method is very useful in rapid screening of the stabilities of new complexes in the gas phase. The influence of the spatial arrangement of the ligands and the type of substituents employed for the complexation were examined. The effect of the preorganization of the diazaperylene on the threshold activation voltages and thus of the relative binding energies of the different complexes are discussed. Density functional theory calculations were used to calculate the optimized structures of the silver complexes and compared with the stabilities of the complexes in the gas phase for the first time.

PH-Dependent Singlet O2 Oxidation Kinetics of Guanine and 9-Methylguanine: An Online Mass Spectrometry and Spectroscopy Study Combined with Theoretical Exploration.

We report a kinetic and mechanistic study on the title reactions, in which 1O2 was generated by the reaction of H2O2 with Cl2 and bubbled into an aqueous solution of guanine and 9-methylguanine (9MG) at different pH values. Oxidation kinetics and product branching ratios were measured using online electrospray ionization mass spectrometry coupled with absorption and emission spectrophotometry, and product structures were determined by collision-induced dissociation (CID) tandem mass spectrometry. Experiments revealed strong pH dependence of the reactions. The oxidation of guanine is noticeable only in basic solution, while the oxidation of 9MG is weak in acidic solution, increases in neutral solution, and becomes intensive in basic solution. 5-Guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) were detected as the major oxidation products of guanine and 9MG, and Sp became dominant in basic solution. A reaction intermediate was captured in mass spectra, and assigned to gem-diol on the basis of CID measurements. This intermediate served as the precursor for the formation of Gh. After taking into account solution compositions at each pH, first-order oxidation rate constants were extracted for individual species: that is, 3.2-3.6 × 107 M-1 s-1 for deprotonated guanine, and 1.2 × 106 and 4.6-4.9 × 107 M-1 s-1 for neutral and deprotonated 9MG, respectively. Guided by approximately spin-projected density-functional-theory-calculated reaction potential energy surfaces, the kinetics for the initial 1O2 addition to guanine and 9MG was evaluated using transition state theory (TST). The comparison between TST modeling and experiment confirms that 1O2 addition is rate-limiting for oxidation, which forms endoperoxide and peroxide intermediates as determined in previous measurements of the same systems in the gas phase.

Collision Induced Dissociation of Benzylpyridinium-Substituted Porphyrins: Towards a Thermometer Scale for Multiply Charged Ions?

We have determined breakdown curves for a range of multiply charged benzylpyridinium-substituted porphyrin cations by collision induced dissociation measurements (CID) as mediated by resonant pulsed radio-frequency (rf) excitation in a helium-filled linear ion trap. Measurements were compared with the predictions of DFT calculations. We find a linear correlation between experimental fragmentation thresholds (in instrumental units of "normalized collision energy") and theoretical dissociation energies, suggesting that these species can be used as calibrants to gauge the fragmentation energetics of closely related systems. We have confirmed this by also studying the fragmentation thresholds of metalloporphyrin-based ions - including multiply negatively charged metalloporphyrin oligomers. Unfortunately, the slope of the linear correlation obtained for benzylpyridinium-substituted porphyrin multications differs significantly from that obtained by us for a set of smaller, singly charged substituted benzylpyridines put forward as "thermometer" ions in previous work. Multiplying the threshold energies in an ad hoc fashion by the ion charge basically reconciles both calibration curves. We conclude that one should use caution when applying small, singly charged benzylpyridines as calibrants to gauge the CID of large, multiply charged ions in ion-trap mass spectrometers. Graphical Abstract.

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%.

Influence of Equilibration Time in Solution on the Inclusion/Exclusion Topology Ratio of Host-Guest Complexes Probed by Ion Mobility and Collision-Induced Dissociation.

Host-guest complexes are formed by the creation of multiple noncovalent bonds between a large molecule (the host) and smaller molecule(s) or ion(s) (the guest(s)). Ion-mobility separation coupled with mass spectrometry nowadays represents an ideal tool to assess whether the host-guest complexes, when transferred to the gas phase upon electrospray ionization, possess an exclusion or inclusion nature. Nevertheless, the influence of the solution conditions on the nature of the observed gas-phase ions is often not considered. In the specific case of inclusion complexes, kinetic considerations must be taken into account beside thermodynamics; the guest ingression within the host cavity can be characterized by slow kinetics, which makes the complexation reaction kinetically driven on the timescale of the experiment. This is particularly the case for the cucurbituril family of macrocyclic host molecules. Herein, we selected para-phenylenediamine and cucurbit[6]uril as a model system to demonstrate, by means of ion mobility and collision-induced dissociation measurements, that the inclusion/exclusion topology ratio varies as a function of the equilibration time in solution prior to the electrospray process.

Distribution of coniferin in differentiating normal and compression woods using MALDI mass spectrometric imaging coupled with osmium tetroxide vapor treatment

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to detect monolignol glucosides in differentiating normal and compression woods of two Japanese softwoods, Chamaecyparis obtusa and Cryptomeria japonica Comparison of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry collision-induced dissociation fragmentation analysis and structural time-of-flight (MALDI-TOF CID-FAST) spectra between coniferin and differentiating xylem also confirmed the presence of coniferin in differentiating xylem. However, as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF CID-FAST spectra of sucrose were similar to those of coniferin, it was difficult to distinguish the distribution of coniferin and sucrose using MALDI-MSI and collision-induced dissociation measurement only. To solve this problem, osmium tetroxide vapor was applied to sections of differentiating xylem. This vapor treatment caused peak shifts corresponding to the introduction of two hydroxyl groups to the C=C double bond in coniferin. The treatment did not cause a peak shift for sucrose, and therefore was effective in distinguishing coniferin and sucrose. Thus, it was found that MALDI-MSI combined with osmium tetroxide vapor treatment is a useful method to detect coniferin in differentiating xylem.

An IMS–IMS threshold method for semi-quantitative determination of activation barriers: Interconversion of proline cis ↔ trans forms in triply protonated bradykinin

Collisional activation of selected conformations by multidimensional ion mobility spectrometry (IMS–IMS), combined with mass spectrometry (MS), is described as a method to determine semi-quantitative activation energies for interconversion of different structures of the nonapeptide bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). This analysis is based on a calibration involving collision-induced dissociation measurements of ions with known dissociation energies (i.e., “thermometer” ions) such as leucine enkephalin, BK, and amino acid–metal cation systems. The energetic barriers between six conformations of [BK+3H]3+ range from 0.23±0.01 to 0.55±0.03eV. Prior results indicate that the major peaks in the IMS distributions correspond to specific combinations of cis and trans configurations of the three proline residues in the peptide sequence. The analysis allows us to directly assess pathways for specific transitions. The combination of structural assignments, experimentally determined barrier heights, onset of the quasi-equilibrium region, and dissociation threshold are used to derive a semi-quantitative potential energy surface for main features of [BK+3H]3+.

Sulfur-34S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34) of Proteomic Changes in Pseudomonas fluorescens during Naphthalene Degradation

The relative quantification of proteins is one of the major techniques used to elucidate physiological reactions. Because it allows one to avoid artifacts due to chemical labeling, the metabolic introduction of heavy isotopes into proteins and peptides is the preferred method for relative quantification. For eukaryotic cells, stable isotope labeling by amino acids in cell culture (SILAC) has become the gold standard and can be readily applied in a vast number of scenarios. In the microbial realm, with its highly versatile metabolic capabilities, SILAC is often not feasible, and the use of other (13)C or (15)N labeled substrates might not be practical. Here, the incorporation of heavy sulfur isotopes is shown to be a useful alternative. We introduce (34)S stable isotope labeling of amino acids for quantification and the corresponding tools required for spectra extraction and disintegration of the isotopic overlaps caused by the small mass shift. As proof of principle, we investigated the proteomic changes related to naphthalene degradation in P. fluorescens ATCC 17483 and uncovered a specific oxidative-stress-like response.

Differential ion mobility separation of isomers for threshold collision-induced dissociation measurements

A triple-quadrupole mass spectrometer adapted for threshold-collision-induced dissociation (TCID) measurements was modified by replacing the standard mass spectrometer front-end with a Fernandez de la Mora-type differential mobility analyzer (DMA). Using a combination of a drift field and an orthogonal laminar gas flow at atmospheric pressure, an ion of a given mobility (DMA voltage) can be isolated from a continuous stream of electrosprayed ions for TCID measurements. Proof-of-concept DMA-TCID experiments using silver complexes of mixtures of ortho–para and cis–trans isomers as well as silver complexes of the individual isomers show excellent agreement in the determined bond dissociation energies (BDEs) between both sets of experiments as well as with theoretical BDEs predicted by density functional theory.

Optimized Fragmentation Conditions for iTRAQ-labeled Phosphopeptides

Protein phosphorylation is an important post-translational modification that plays a regulatory role within numerous biological processes. The simultaneous identification, localization, and quantification of phosphorylated proteins is vital for understanding this dynamic control mechanism. The application of isobaric labeling strategies, for example, iTRAQ, for quantitative phosphopeptide analysis requires simultaneous monitoring of peptide backbone fragmentation, loss of phosphoryl moieties, and the cleavage of isobaric labeling reporter ions. In the present study, we have examined MS/MS fragmentation modes available in the Orbitrap Velos MS (collision induced dissociation (CID), CID plus multistage activation, and higher energy collision dissociation (HCD)), for their ability to generate ions required for simultaneous quantification and identification of iTRAQ labeled phosphopeptides in a semicomplex (12) and a complex (131) phosphopeptide mix. The required normalized collision energies for quantification and identification of iTRAQ-labeled phosphopeptides require a compromise between the optimal parameters for each aspect. Here, we were able to determine an optimized MS/MS measurement protocol that involves CID measurement in ion trap for identification followed by HCD measurement for parallel identification and quantification that satisfies the time requirements for LC-MS/MS experiments.

Role of methylation on the thermochemistry of alkali metal cation complexes of amino acids: N-methyl proline

Quantitative thermodynamic information is obtained from the study of the gas-phase interactions of the alkali metal cation complexes of N-methyl proline (NMP) with Xe using a guided ion beam tandem mass spectrometer (GIBMS). Absolute bond dissociation energies (BDEs) of M+=Li+, Na+, K+, and Rb+ to NMP are determined experimentally from threshold collision-induced dissociation (TCID) measurements of the M+(NMP) complexes. Analysis of their kinetic energy cross sections provide the 0K bond enthalpies after accounting for unimolecular decay rates, internal energy of reactant ions, and multiple ion-molecule collisions. Quantum chemical calculations of the M+(NMP) BDEs are found to be in good agreement with the experimental values, establishing that the zwitterionic form is the lowest energy structure for all the metal ion complexes. Compared to M+(Pro) BDEs, the metal binding in these zwitterions is slightly enhanced by the CH3 group on the ring nitrogen, presumably a result of an inductive effect and its higher polarizability. More profound consequences of the methyl group emerge in the charge-solvated conformers calculated for M+(NMP) where it directs multiple conformations of the pyrrolidine ring. This is unlike the ring puckering phenomenon seen in M+(Pro) complexes, where fewer conformations are found, apparently because inversion at the nitrogen center is more facile.

Hydration Energies of Aromatic Ions in the Gas Phase

The hydration energies of aromatic ions, measured by using energy-resolved collision-induced dissociation measurements, are reported. The hydration energies of protonated acetophenone, aniline, anisole, benzene, benzonitrile, phenol, and toluene are 0.67 ± 0.04, 0.62 ± 0.04, 0.86 ± 0.04, 0.46 ± 0.06, 0.84 ± 0.04, 0.75 ± 0.07, and 0.42 ± 0.04 eV, respectively. The measured values are in good agreement with those predicted by using coupled cluster theory, provided the proper geometries of the ions and sufficient basis set were used.