MALDI-TOF/TOF CID study of 4-alkyl-substituted polystyrene fragmentation reactions

MALDI-TOF/TOF CID experiments were conducted on a variety of hydrogen-terminated poly(4-methylstyrene), hydroxylated poly(t-butylstyrene), and polystyrene precursor ions: n = 10, 15, 20, 25, and 30, where the number of repeat units n corresponds to the oligomer mass number. The influences of structure, molecular weight, and effective collision kinetic energy on degradation mechanisms were examined to test the generality of our multi-chain fragmentation model developed for polystyrene. Each depolymerization mechanism is presented in detail with experimental and computational data to justify/rationalize its occurrence and effective kinetic energy dependence. These processes show the complex interrelationship between the various pathways along with preferred production of secondary radicals, which suppresses the appearance of primary radicals. Additionally, Py-GC/MS experimental data are presented, for comparison of the multimolecular free radical reactions in pyrolysis with the unimolecular fragmentation reactions of MS/MS. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studying disulfide bond rearrangement by MALDI-RTOF PSD and MALDI-TOF/RTOF high-energy CID (20 keV) experiments of peptides derived from ammodytoxins

Ammodytoxins (Atxs) are presynaptically neurotoxic phospholipases present in Vipera ammodytes ammodytes snake venom. Atxs show a high sequence homology and contain 14 cysteines which form seven biologically relevant disulfide bridges-connecting non-neighboring cysteines. Formic acid cleavage was performed to confirm protein sequences by MALDI RTOF MS and resulted in 95.6% sequence coverage exhibiting only few formylations. Cysteine-containing peptides showed adjacent signals 2 and/or 4 Da lower (according to the number of cysteines present in the peptide) than the theoretical molecular weight indicating disulfide bridge rearrangement. Post-source decay (PSD) and high-energy collision-induced dissociation (CID) at 20 keV experiments showed fragmentation pattern unique for the reduced, thiol group containing and the oxidized, disulfide bridge harboring peptides. Besides typical low-energy fragment ions observed during PSD experiments (a-, b-, y-type ions), additional high-energy fragment ions (c-, x-, w-, d-type and internal fragments) of significant intensity were generated during fragmentation at 20 keV. In the case of charge directing N- and C-termini, x- and w-type ions were also observed during PSD. Good and up to complete sequence coverage was achieved for all studied peptides from Atxs in the case of high-energy CID, whereas PSD lacked information particularly for larger peptides.     

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.     

Oxygenated triacylglycerols of the lipids ofOnopordum acanthium seeds

The results on the structure and composition of the epoxyacyl-, hydroxyacyl-, and epoxyacylhydroxyacylacylglycerols ofOnopordum acanthium L. seeds obtained by enzymatic hydrolysis have been supplemented with the aid of mass spectrometry. The desirability of using a combination of the two methods for these purposes has been shown.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 656–670, September–October, 1993.     

Theoretical study of alpha/beta-alanine and their protonated/alkali metal cationized complexes

Density functional theory has been employed to model the structure and the relative stabilities of alpha/beta-alanine conformers and their protonated and alkali metal cationized complexes. In general, we find that the behavior of the beta-alanine (beta-Ala) system is quite similar to that of alpha-alanine (alpha-Ala). However, the presence of the methylene group (-CH2-) at the beta position in beta-Ala leads to a few key differences. First, the intramolecular hydrogen bonding patterns are different between free alpha- and beta-Ala. Second, the stability of zwitterionic species (in either the free ligand or alkali metal cationized complexes) is often enhanced in beta-Ala. Third, the preferred mode of alkali metal cation (M+) binding may also differ in alpha- and beta-Ala. Natural energy decomposition analysis has been applied here to gain further insight into the effects of the ligand, cation size, and mode of binding on the nature of interaction in these M+-Ala complexes.     

Pyrolysis-GC-FTIR for structural elucidation of aquatic humic substances

Substructure components of various aquatic humic substances were investigated by a coupled pyrolysis — gas chromatography — Fourier-transform infrared spectroscopy (Py-GC-FTIR) procedure. The humic substances studied gave similar pyrolysis products, but in varying proportions. Many of the pyrolysis products (e.g. methanol, acetone, alkylbenzenes, cyclopentane, aliphatic and aromatic organic acids, acetamide, pyrrole and phenols) could be identified by their FTIR spectra using a digital library for automatic comparison. Some of the compounds are related to lignin fragments which form a large part of the humic substances investigated. Other products give hints to the involvement of tetrapyrroles, fatty acids, furanoses and amino compounds in the structure of humic macromolecules.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Electrospray ionization multiple-stage linear ion-trap mass spectrometry for structural elucidation of triacylglycerols: Assignment of fatty acyl groups on the glycerol backbone and location of double bonds

Linear ion-trap multiple-stage mass spectrometric approach (MS ⁿ ) towards nearly complete structural elucidation of triacylglycerol (TAG) including (1) assignment the fatty acid substituents on the glycerol backbone and (2) location of the double bond(s) on the unsaturated fatty acyl groups is reported. The characterization is established by the findings that MS² on the [M+Li]⁺ ions of TAG yields more abundant ions reflecting losses of the outer fatty acid substituents either as free acids (i.e., [M+Li-R₁CO₂H]⁺ and [M+Li-R₃CO₂H]⁺ ions) or as lithium salts (i.e., [M+Li-R₁CO₂Li]⁺ and [M+Li-R₃CO₂Li]⁺ ions) than the ions reflecting the similar losses of the inner fatty acid substituent (i.e., [M+Li-R₂CO₂Li]⁺ and [M+Li-R₂CO₂Li]⁺ ions). Further dissociation (MS³) of [M+Li-R _n CO₂H]⁺ (n=1, 2, or 3) gives rise to the ion series locating the double bonds along the fatty acid chain. These ions arise from charge-remote fragmentations involving β-cleavage with γ-H shift, analogous to those seen for the unsaturated long-chain fatty acids characterized as initiated ions. Significant differences in abundances in the ion pairs reflecting the additional losses of the fatty acid moieties, respectively, were also seen in the MS³ spectra of the [M+Li-R _n CO₂H]⁺ and [M+Li-R _n CO₂Li]⁺ ions, leading to confirmation of the fatty acid substituents on the glycerol backbone. MS ⁿ on the [M+Na]⁺ and [M+NH₄]⁺ adduct ions also affords location of fatty acid substituents on the glycerol backbone, but not the position of the double bond(s) along the fatty acid chain. Unique ions from internal losses of the glycerol residues were seen in the MS³ spectra of [M+Alk-R _n CO₂H]⁺ (n=1, 2, 3) and of [M+Alk-R _n CO₂Alk]⁺ (Alk=Li, Na, NH₄; n=1, 3). They are signature ions for glycerides and the pathways leading to their formation may involve rearrangements.     

The structural elucidation of two new artificial steroidal saponins

Two novel steroidal saponins(timosaponin BⅢ-a,timosaponin BⅢ-b) together with a known steroidal saponin(timosaponin BⅡ-b) were prepared by the dilute acid hydrolysis of timosaponin BⅢ.Their structures were elucidated by means of 1D,2D NMR and TOF-MS spectral analysis.     

Benzamidinium d-glucuronate: Spectroscopic and structural elucidation

Benzamidinium d-glucuronate (1) crystallizes in the orthorhombic space group P2₁2₁2₁ and exhibits a 3 D network with molecules linked by moderate intermolecular hydrogen bonds (HNH…O_(solvent) 2.993 Å, HNH…OCO 2.894 Å, HNH…O_(cycle) 2.844 Å, OH…NH₂ 2.931 Å, OH…O_(solvent) 2.894, 2.924 and 2.715 Å (stronger)) with participation of cations, anions and solvent molecules. The IR-band assignment of carbohydrate moieties is elucidated by a comparison between the types and bond lengths of intermolecular interactions with participation of OH groups in d-glucuronate and linear polarized IR-(IR-LD) spectroscopic data. Experimental results are supported by theoretical ab initio calculations of benzamidinium cation and d-glucuronate anion.     

The propensity rules re-examined: Evidence for a long-lived complex in alkali diatomic-rare-gas collisions

When ΔJ =±1 collision-induced transitions in Π states of excited alkali diatomics are examined over a wide range of pressures several curious phenomena are observed. With the heavier rare gases we find that the ± propensities reverse as pressure increases, and the circular polarisation of the parent line first decreases, with increasing pressure and then begins to rise, the change-over occurring at roughly the same pressure as the reversal ΔJ =±1 collision propensities. A recently proposed theoretical model involving the formation of a long-lived atom—diatomic collision complex or Feschbach resonance is invoked to explain these unusual experimental results.     

Energetics and structural elucidation of mechanisms for gas phase H/D exchange of protonated peptides

Hydrogen/deuterium exchange reactions involving protonated triglycine and deuterated ammonia (ND(3)) have been examined in the gas phase using a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Ab initio and density functional theory (DFT) calculations have been carried out to model the exchanges and to obtain energetics and vibrational frequencies for molecules involved in the proposed exchange mechanisms. Structural optimization and frequency calculations have been performed at the B3LYP level of theory with the 6-311+G(d,p) basis set. Transition states have been calculated at the same level of theory and basis set as above using the QST2 and QST3 methods. Single-point energy calculations have been performed at the MP2/6-311+G(d,p) level. Six labile sites of protonated triglycine were found to undergo H/D exchange. Of these six labile hydrogens, two are amide, three are ammonium, and one is carboxyl. Detailed mechanisms for each of these transfers are proposed. Qualitative onium ion and tautomer mechanisms for the exchanges of ammonium and amide hydrogens, respectively, using semiempirical calculations were suggested in previous studies by Beauchamp et al. As shown by the current ab initio and DFT calculations completed during this study, the mechanisms proposed in that study are notionally correct; however, the tautomer mechanisms are shown here to be the result of the fact that a second stable isomer of protonated triglycine exists in which the amide1 carbonyl oxygen is protonated. The exchange of the carboxyl hydrogen is found to proceed via a transition state resembling an ammonium ion interacting with a carboxylate moiety via two hydrogen bonds. The current work thus provides significant mechanistic and structural detail for a considerably more in-depth understanding of the processes involved in gas phase H/D exchange of peptides.     

Low‐energy collision‐induced dissociation (low‐energy CID), collision‐induced dissociation (CID), and higher energy collision dissociation (HCD) mass spectrometry for structural elucidation of saccharides and clarification of their dissolution mechanism in DMAc/LiCl

The dissolution mechanism of oligosaccharides in N,N‐dimethylacetamide/lithium chloride (DMAc/LiCl), a solvent used for cellulose dissolution, and the capabilities of low‐energy collision‐induced dissociation (low‐energy CID), collision‐induced dissociation (CID), and higher energy collision dissociation (HCD) for structural analysis of carbohydrates were investigated. Comparing the spectra obtained using 3 techniques shows that, generally, when working with monolithiated sugars, CID spectra provide more structurally informative fragments, and glycosidic bond cleavage is the main pathway. However, when working with dilithiated sugars, HCD spectra can be more informative providing predominately cross‐ring cleavage fragments. This is because HCD is a nonresonant activation technique, and it allows a higher amount of energy to be deposited in a short time, giving access to more endothermic decomposition pathways as well as consecutive fragmentations. The difference in preferred dissociation pathways of monolithiated and dilithiated sugars indicates that the presence of the second lithium strongly influences the relative rate constants for cross‐ring cleavages vs glycosidic bond cleavages, and disfavors the latter. Regarding the dissolution mechanism of sugars in DMAc/LiCl, CID and HCD experiments on dilithiated and trilithiated sugars reveal that intensities of product ions containing 2 Li⁺ or 3 Li⁺, respectively, are higher than those bearing only 1 Li⁺. In addition, comparing the fragmentation spectra (both HCD and CID) of LiCl‐adducted lithiated sugar and NaCl‐adducted sodiated sugar shows that while, in the latter case, loss of NaCl is dominant, in the former case, loss of HCl occurs preferentially. The compiled evidence implies that there is a strong and direct interaction between lithium and the saccharide during the dissolution process in the DMAc/LiCl solvent system.     

A new charge-associated mechanism to account for the production of fragment ions in the high-energy CID spectra of fatty acids

A new mechanism, termed a charge-assisted process, is proposed as an additional mechanism to the charge-remote process to account for ions of the [M - CnH2n+2] series found in the positive and negative high energy CID spectra of fatty acids and related compounds when ionized as closed-shell ([M - H]- or [M + X]+) species. The new mechanism is based on that commonly invoked to account for similar ions in the electron-impact spectra of derivatized fatty acids whereby the positive charge on the derivative abstracts a hydrogen atom from various positions of the alkyl chain to leave a radical that initiates a radical-induced cleavage of the chain. It is proposed that in the high energy CID spectra of closed-shell ions, similar hydrogen migrations occur but unpairing of electrons is avoided by charge transfer to the alkyl chain. This charge then initiates a concerted cleavage of the chain to give an allylic carbonium (positive ion spectrum) or carbanion (negative ion spectrum). The mechanism avoids the need to involve radicals or loss of hydrogen atoms from even-electron (closed shell) ions and provides a driving force for the reaction, namely, the formation of ions with a stabilized charge. An extension of the mechanism is also proposed to account for the formation of odd-electron ions from these compounds. The charge-assisted mechanism does not rule out the occurrence of other mechanisms that have been accepted for many years but provides an alternative process that can account for some spectral features which were difficult to explain earlier.     

Absolute complex formation cross sections for alkali—alkali halide collisions: a Monte Carlo trajectory study

A classical Monte Carlo trajectory study has been performed, where the potential energy function used is an ideal dipole potential with its center displaced from the center of mass. The potential shape depends on two parameters. For one choice of parameters, the theoretical Roach and Child surface is well approximated at large and intermediate distances. Several other choices of parameter values have also been used. Complex formation cross sections and angular distributions at the centrifugal barrier have been determined, and a comparison with the theoretical quantities for a spherically symmetric potential is made. The previously proposed mechanisms, assumed to explain the experimentally found low values of σ_c and the quantity of Γ, have not been found to be significant. A dynamical picture is used to interpret the present results. It also provides an interpretation of the previous experimental results. In this picture rotational energy transfer at the barrier is of great importance.     

The synthesis and first full structural elucidation of a benzotriazole azo dye

Although azo dyes containing benzotriazole are of interest as substrates for surface enhanced resonance Raman scattering, SERRS, little is known of their molecular structure due to their poor crystal growth properties. We recently synthesised a highly crystalline dihydroquinoline via an unusual condensation reaction and we report herein that the azo‐benzotriazole dye subsequently formed was sufficiently crystalline to allow structural elucidation using synchroton radiation. It was found that this benzotriazole dye exists as the unexpected 6‐isomer and the structure of the dihydroquinoline moiety changes markedly on coupling due to increased delocalisation.     

Comparison of laser-induced dissociation and high-energy collision-induced dissociation using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) for peptide and protein identification

The fragmentation of peptides under laser-induced dissociation (LID) as well as high-energy collision-induced dissociation (CID) conditions has been investigated. The effect of the different fragmentation mechanisms on the formation of specific fragment ion types and the usability of the resulting spectra, e.g. for high-throughput protein identification, has been evaluated. Also, basic investigations on the influence of the matrix, as well as laser fluence, on the fragment ion formation and the consequences in the spectral appearance are discussed. The preconditions for obtaining 'pure' CID spectra on matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) instruments are evaluated and discussed as well as the differences between LID and CID in the resulting fragment ion types. While containing a wealth of information due to additional fragment ions in comparison with LID, CID spectra are significantly more complex than LID spectra and, due to different fragmentation patterns, the CID spectra are of limited use for protein identification, even under optimized parameter settings, due to significantly lower scores for the individual spectra. Conditions for optimal results regarding protein identification using MALDI-TOF/TOF instruments have been evaluated. For database searches using tandem mass spectrometric data, the use of LID as fragmentation technique in combination with parameter settings supporting the use of internal fragment ions turned out to yield the optimal results.     

A comparison of accurate mass techniques for the structural elucidation of fluconazole

Mass spectrometry plays a major role in the structural elucidation and characterisation of drug candidates and related substances. Accurate mass data allow the mathematical prediction of molecular formula of both precursor and fragment ions. In this paper, a comparison of the accurate mass data obtained for the fragmentation of fluconazole, an antifungal drug, by three different methods is made: electron ionisation (EI) using a magnetic sector instrument; electrospray ionisation (ES) using a Fourier transform ion cyclotron mass spectrometer (FTICRMS); and ES using a quadrupole-time-of-flight mass spectrometer (Q-ToF). It is clear from the data obtained that mass accuracy is not simply a function of instrument resolution. The subtle differences observed between collisionally activated dissociation (CAD) and sustained off-resonance collisionally activated dissociation (SORI-CAD) spectra are explained as a consequence of the excitation process. The advantages and disadvantages of the three techniques are discussed within the context of structural elucidation.     

Calculated NMR as a tool for structural elucidation of jungianol and mutisianthol

Theoretical methods were used for the correct structure assignments of the natural products jungianol and mutisianthol. A three stage protocol for the calculation was used: a conformational search using molecular mechanics (MM3), a DFT (B3LYP) structure optimization and ab initio (HF/GIAO) or DFT (B3LYP/GIAO) calculation of magnetic properties.