Acid‐catalysed glucose dehydration in the gas phase: a mass spectrometric approach

Understanding on a molecular level the acid‐catalysed decomposition of the sugar monomers from hemicellulose and cellulose (e.g. glucose, xylose), the main constituent of lignocellulosic biomass is very important to increase selectivity and reaction yields in solution, key steps for the development of a sustainable renewable industry. In this work we reported a gas‐phase study performed by electrospray triple quadrupole mass spectrometry on the dehydration mechanism of d ‐glucose. In the gas phase, reactant ions corresponding to protonated d ‐glucose were obtained in the ESI source and were allowed to undergo collisionally activated decomposition (CAD) into the quadrupole collision cell. The CAD mass spectrum of protonated d ‐glucose is characterized by the presence of ionic dehydrated daughter ion (ionic intermediates and products), which were structurally characterized by their fragmentation patterns. In the gas phase d ‐glucose dehydration does not lead to the formation of protonated 5‐hydroxymethyl‐2‐furaldehyde, but to a mixed population of m/z 127 isomeric ions. To elucidate the d ‐glucose dehydration mechanism, 3‐O‐methyl‐d ‐glucose was also submitted to the mass spectrometric study; the results suggest that the C3 hydroxyl group plays a key role in the reaction mechanism. Furthermore, protonated levulinic acid was found to be formed from the monodehydrated d ‐glucose ionic intermediate, an alternative pathway other than the known route consisting of 5‐hydroxymethyl‐2‐furaldehyde double hydration. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination and enhancement of stereospecific decompositions via triple mass spectrometry: Formation of untypical protonated precursor molecules

A tetraquadrupole mass spectrometer with consecutive surface-induced dissociation/collisionally activated dissociation (SID/CAD) capability has been used to investigate the decompositional behaviour of bifunctional terpenes. SID and CAD yield similar daughter-ion spectra of protonated molecules generated by ammonia chemical ionization. These collision mass spectra of MH⁺ contain diagnostic daughter ions which can be used to distinguish diastereomeric terpenols. Pronounced stereochemical effects underlying specific decompositions of the ammonium adduct and protonated molecule forms of the bifunctional terpenes have been attributed to the formation of protonated molecules of lower stability produced via decomposition of [M + NH₄]⁺. Evidence supporting the existence of such unusual protonated molecules formed via collision is given in the grand-daughter spectra of [M + NH₄]⁺. Triple mass Spectrometry is shown to promote the Stereospecific formation and subsequent diagnostic decomposition of these singular protonated forms, thus improving the ability to differentiate the diastereomers.     

Characterization of the Dissociation Behavior of Gas-Phase Protonated and Methylated Lactones

The dissociation behavior of gas-phase protonated and methylated four-, five-, six-, and seven-membered ring lactones, some with methyl substituents in various positions, has been characterized by using a quadrupole ion trap mass spectrometer and a triple quadrupole mass spectrometer. The energy dependence of collisionally activated dissociation pathways was determined by energy-resolved mass spectrometry, and the dissociation behavior of the various protonated lactones was compared to that observed for protonated cyclic ketones and ethers of analogous ring size. The protonated cyclic ethers and ketones predominantly dissociated via dehydration, whereas the protonated lactones dissociated via losses of an alkene, ketene, and water. The dissociation behavior of the gas-phase methylated lactones formed from ion/molecule reactions with dimethyl ether ions was compared to the collisionally activated dissociation behavior of isomeric protonated methyl-substituted lactones. The methylation experiments indicated that the gas-phase addition of a methyl group may dramatically alter the favored dissociation pathways when compared to the simple protonated ions.     

Decomposition of protonated threonine, its stereoisomers, and its homologues in the gas phase: evidence for internal backside displacement

Protonated threonine and its allo diastereomer exhibit different proportions of collisionally activated dissociation (CAD) product ions. N-Methylation attenuates these differences. Water loss from protonated allo-threonine gives protonated trans-3-methylaziridinecarboxylic acid via an internal S(N)2 pathway, rather than protonated vinylglycine.     

Further studies on the origins of asymmetric charge partitioning in protein homodimers

Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of alpha-lactalbumin homodimers with 15+ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8+/7+. In contrast, dissociation of the 15+ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11+ charges, and the oxidized molecule carries away 4+ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17+ charge state undergoes symmetric charge partitioning whereas dissociation of the 13+ is asymmetric. Reduction of the charge state of dimers with 17+ charges to 13+ via gas-phase proton transfer and subsequent dissociation of the mass selected 13+ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13+ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments.     

Hydrogen-bonding interactions in gas-phase polyether/ammonium ion complexes

Hydrogen bonds are among the most important interactions involved in selective complexation in host-guest chemistry. In this study a variety of hydrogen-bonded crown ether/ammonium ion complexes are generated in the gas phase by association reactions between an amine substrate and a polyether, one of which is initially protonated, and stabilized by many collisions in the chemical ionlzation source of a triple quadrupole mass spectrometer or in a quadrupole ion trap. The nature of the hydrogen-bonding interactions of the ion complexes are evaluated by comparison of their collision-activated dissociation spectra. After collisional activation, those complexes that are weakly bound dissociate to form intact protonated polyether molecules and/or ammonium ions by simple cleavages of the hydrogen-bond association interactions. In contrast, those complexes strongly bound by multiple hydrogen bonds dissociate not only to the protonated polyether and/or ammonium ions but also by extensive covalent bond cleavage of the protonated ether skeleton.This latter type of dissociation behavior suggests that the polyether/ammonium ion complexes may be sufficiently strongly bound that surpassing the high barrier to decomposition results in formation of internally excited polyether molecules that may then undergo subsequent fragmentation by skeletal cleavages. Moreover, complexes involving multiple hydrogen bonds may have slower dissociation kinetics, allowing competition from fast dissociation processes that have substantial energy barriers.     

Top-Down Mass Spectrometry of Supercharged Native Protein-Ligand Complexes

Tandem mass spectrometry (MS/MS) of intact, noncovalently-bound protein-ligand complexes can yield structural information on the site of ligand binding. Fourier transform ion cyclotron resonance (FT-ICR) top-down MS of the 29 kDa carbonic anhydrase-zinc complex and adenylate kinase bound to adenosine triphosphate (ATP) with collisionally activated dissociation (CAD) and/or electron capture dissociation (ECD) generates product ions that retain the ligand and their identities are consistent with the solution phase structure. Increasing gas phase protein charging from electrospray ionization (ESI) by the addition of supercharging reagents, such as m-nitrobenzyl alcohol and sulfolane, to the protein analyte solution improves the capability of MS/MS to generate holo-product ions. Top-down proteomics for protein sequencing can be enhanced by increasing analyte charging.     

High resolution and tandem fourier-transform mass spectrometry with californium-252 plasma desorption

For ions formed by plasma desorption (PD) in a Fourier-transform mass spectrometer, high resolution measurements are demonstrated, such as 65,000 (FWHH) for the protonated molecular ion of gramicidin S (MW 1140.7). Resolution is substantially improved by delaying measurements until a significant ion concentration has built up in the cell, and by collisionally deactivating the orbital kinetic energy of the ions. This also makes the ions available for subsequent dissociation steps, so that tandem mass spectrometry can be demonstrated for PD ions. With this for larger ions, collisionally activated dissociation (CAD) is affected with> 85% efficiency. The CAD spectra of (M + Na)⁺ and of fragment ions from the PD of gramicidin S provide structurally useful information.     

The effective temperature of Peptide ions dissociated by sustained off-resonance irradiation collisional activation in fourier transform mass spectrometry

A method for determining the internal energy of biomolecule ions activated by collisions is demonstrated. The dissociation kinetics of protonated leucine enkephalin and doubly protonated bradykinin were measured using sustained off-resonance irradiation (SORI) collisionally activated dissociation (CAD) in a Fourier transform mass spectrometer. Dissociation rate constants are obtained from these kinetic data. In combination with Arrhenius parameters measured with blackbody infrared radiative dissociation, the "effective" temperatures of these ions are obtained. Effects of excitation voltage and frequency and the ion cell pressure were investigated. With typical SORI-CAD experimental conditions, the effective temperatures of these peptide ions range between 200 and 400 degrees C. Higher temperatures can be easily obtained for ions that require more internal energy to dissociate. The effective temperatures of both protonated leucine enkephalin and doubly protonated bradykinin measured with the same experimental conditions are similar. Effective temperatures for protonated leucine enkephalin can also be obtained from the branching ratio of the b(4) and (M + H - H(2)O)(+) pathways. Values obtained from this method are in good agreement with those obtained from the overall dissociation rate constants. Protonated leucine enkephalin is an excellent "thermometer" ion and should be well suited to establishing effective temperatures of ions activated by other dissociation techniques, such as infrared photodissociation, as well as ionization methods, such as matrix assisted laser desorption/ionization.     

Multi-stage collisionally-activated decomposition in an ion trap for identification of sequences, structures and bn --> bn-1 fragmentation pathways of protonated cyclic peptides

Cyclic penta-, hexa- and heptapeptides have been designed, synthesized and their fragmentations induced by multistage tandem mass spectrometry have been studied. Under low-energy collisionally activated decomposition (CAD), the protonated cyclic peptides mainly dissociate via ring opening pathways and the corresponding bn --> bn-1 pathways to form several sets of b ions as oxazolone rings (and b1 ions as aziridinone rings). Through repeated observation of these b ions in multistep CAD experiments, accurate sequencing and head-to-tail ring structure of cyclic peptides can be determined. The mistaken assignments of these b ions can be avoided by this sequencing method. Semiempirical molecular orbital calculations have been utilized to provide insight into the proposed dissociation mechanism. In addition, for cyclic peptides that include an Asn residue, the nitrogen of the Asn side chain is observed to be preferentially protonated, which can induce a unique ring-opening pathway with a loss of ammonia that competes with the conventional ring opening pathway.     

Thermal dissociation of the protein homodimer ecotin in the gas phase

The influence of charge on the thermal dissociation of gaseous, protonated, homodimeric, protein ecotin ions produced by nanoflow electrospray ionization (nanoES) was investigated using the blackbody infrared radiative dissociation technique. Dissociation of the protonated dimer, (E₂ + nH)ⁿ⁺ E₂ⁿ⁺ where n = 14–17, into pairs of monomer ions is the dominant reaction at temperatures from 126 to 175 °C. The monomer pair corresponding to the most symmetric charge distribution is preferred, although 50–60% of the monomer product ions correspond to an asymmetric partitioning of charge. The relative abundance of the different monomer ion pairs produced from E₂¹⁴⁺, E₂¹⁵⁺, and E₂¹⁶⁺ depends on reaction time, with the more symmetric charge distribution pair dominating at longer times. The relative yield of monomer ions observed late in the reaction is independent of temperature indicating that proton transfer between the monomers does not occur during dissociation and that the different monomer ion pairs are formed from dimer ions which differ in the distribution of charge between the monomers. For E₂¹⁷⁺, the yield of monomer ions is independent of reaction time but does exhibit slight temperature dependence, with higher temperatures favoring the monomers corresponding to most symmetric charge distribution. The charge distribution in the E₂¹⁵⁺ and E₂¹⁶⁺ dimer ions influences the dissociation kinetics, with the more asymmetric distribution resulting in greater reactivity. In contrast, the charge distribution has no measurable effect on the dissociation kinetics and energetics of the E₂¹⁷⁺ dimer.     

Chiral enrichment of serine via formation,dissociation, and soft-landing of octameric cluster ions

Chiral enrichment of serine is achieved in experiments that involve formation of serine octamers starting from non-racemic serine solutions. Serine octamers were generated by means of electrospray and sonic spray ionization of aqueous solutions of d(3)-L-serine (108 Da) and D-serine (105 Da) having different molar ratios of enantiomers. A cyclic process involving the formation of chirally-enriched octameric cluster ions and their dissociation, viz. Ser(1) --> Ser(8) --> Ser(1), allows serine monomers to be regenerated with increased enantiomeric excess as shown in two types of experiments: (1) Chiral enrichment in serine was observed in MS/MS/MS experiments in a quadrupole ion trap in which the entire distribution of serine octamers formed from non-racemic solutions was isolated, collisionally activated, and fragmented. Monomeric serine was regenerated with increased enantiomeric excess upon dissociation of octamers when compared with the enantiomeric composition of the original solution. (2) Chiral enrichment was observed in the products of soft-landing of mass-selected protonated serine octamers. These ions were generated by means of electrospray or sonic spray ionization, mass selected, and collected on a gold surface using ion soft-landing. Chiral enrichment of the soft-landed serine was established by redissolving the recovered material and comparing the intensities of protonated molecular ions of d(3)-L-serine and D-serine after APCI-MS analysis. Both of these experiments showed comparable results, suggesting that formation of serine octamers depends only on the enantiomeric composition of the serine solution and that the magnitude of the chiral preference is intrinsic to octamers formed from solutions of given chiral composition.     

Self-Assembling of cytosine nucleoside into triply-bound dimers in acid Media. A comprehensive evaluation of proton-bound pyrimidine nucleosides by electrospray tandem mass spectrometry,X-rays diffractometry,and theoretical calculations

Electrospray tandem mass spectrometry (ESI-MS/MS) is used to evaluate the assembling of cytosine and thymine nucleosides in the gas phase, through the formation of hydrogen bonded supermolecules. Mixtures of cytidine analogues and homologues deliver in the gas phase proton-bound heterodimers stabilized by multiple interactions, as proven by the kinetics of their dissociation into the corresponding protonated monomers. Theoretical calculations, performed on initial structures of methylcytosine homodimers available in the literature, converged to a minimized structure whereby the two pyrimidine rings interact through the formation of three hydrogen bonds of similar energy. The crystallographic data here reported show the equivalency of the two interacting pyrimidines which is attributable to the presence of an inversion center. Thymine and uracil pyrimidyl nucleosides form, by ESI, gaseous proton-bound dimers. The kinetic of their dissociation into the related protonated monomers shows that the nucleobases are weekly interacting through a single hydrogen bond. The minimized structure of the protonated heterodimer formed by thymine and N-1-methylthymine confirmed the existence of mainly one hydrogen bond which links the two nucleobases through the O4 oxygens. No crystallographic data exists on thymine proton-bound species, nor have we been able to obtain these aggregates in the solid phase. The gaseous phase, under high vacuum conditions, seems therefore a suitable environment where vanishing structures produced by ESI can be studied with a good degree of approximation.     

Mass spectral characterization of tetracyclines by electrospray ionization,H/D exchange,and multiple stage mass spectrometry

Electrospray ionization (ESI) and collisionally induced dissociation (CID) mass spectra were obtained for five tetracyclines and the corresponding compounds in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The number of labile hydrogens, x, could easily be determined from a comparison of ESI spectra obtained with N2 and with ND3 as the nebulizer gas. CID mass spectra were obtained for [M + H]+ and [M - H]- ions and the exchanged analogs, [M(Dx) + D]+ and [M(Dx) - D]- , and produced by ESI using a Sciex API-III(plus) and a Finnigan LCQ ion trap mass spectrometer. Compositions of product ions and mechanisms of decomposition were determined by comparison of the MS(N) spectra of the un-deuterated and deuterated species. Protonated tetracyclines dissociate initially by loss of H2O (D2O) and NH3 (ND3) if there is a tertiary OH at C-6. The loss of H2O (D2O) is the lower energy process. Tetracyclines without the tertiary OH at C-6 lose only NH3 (ND3) initially. MSN experiments showed easily understandable losses of HDO, HN(CH3)2, CH3 - N=CH2, and CO from fragment ions. The major fragment ions do not come from cleavage reactions of the species protonated at the most basic site. Deprotonated tetracyclines had similar CID spectra, with less fragmentation than those observed for the protonated tetracyclines. The lowest energy decomposition paths for the deprotonated tetracyclines are the competitive loss of NH3 (ND3) or HNCO (DNCO). Product ions appear to be formed by charge remote decompositions of species de-protonated at the C-10 phenol.     

Observation of self-ion-molecule reactions during collisionally activated dissociation in an ion-trap mass spectrometer

This paper describes the formation of protonated molecules ([M + H]+) and adduct ions by self-ion-molecule reactions (SIMR) during collisionally activated decomposition (CAD) of methyne addition ions ([M + CH]+) produced from chemical ionization (CI) or SIMR in both an external and internal source ion-trap mass spectrometer (ITMS). The CAD results for the methyne addition ions of dopamine produced from both SIMR and dimethyl ether CI undertaken in the external and internal source ITMS were compared in order to prove the occurrence of SIMR during CAD processes. Compared with the external source ITMS, the internal source ITMS is much more easily applicable to this type of reaction owing to the large population of neutral analytes present in the trap.     

MS<Superscript>n</Superscript> characterization of protonated cyclic peptides and metal complexes

MSⁿ experiments involving low energy collisionally activated dissociation (CAD) in a quadrupole ion trap were used to characterize the fragmentation of alkali, alkaline earth and transition metal complexes of five cyclic peptides, and the results were compared with those obtained for protonated cyclic peptides. Complexes with metal ions produced enhanced abundances of the most diagnostic fragments for elucidating the primary structures. For cyclosporin A, nickel and lithium complexes gave additional sequence information compared with the protonated peptide. For depsipeptides, sodium and lead complexes were superior to the protonated peptide or other metal complexes for sequencing residues, and CAD of the lead complexes led to preferential cleavage of two residues at a time. For cyclic lipopeptides, complexes with silver, nickel and strontium ions provided enhanced abundances of key fragment ions.     

Decompositions of cationized heterodimers of amino acids in relation to charge location in peptide ions

The unimolecular decompositions of protonated heterodimers of native and derivatized amino acids to yield the protonated monomers were studied as a guide to charge location in peptide ions. Analyses using a hybrid instrument of BEqQ geometry demonstrated the advantages (with respect to mass resolution, sensitivityr reproducibility, and the elimination of extraneous signals) of the detection of product ions formed in the radiofrequency-only quadrupole region (q) rather than in the field-free region between Band E. Conversion of arginine to dimethylpyrimidylomithine (DMPO) reduced the proton affinity, as evidenced by the decomposition of the protonated arginine/DMPO heterodimer. Conversion of cysteine to pyridylethylcysteine enhanced the proton affinity. Application of these derivatization procedures to peptides resulted in changes in the observed fragmentations of the protonated precursors consistent with the predicted modifications in charge location. Unimolecular decomposition of the protonated dimer composed of glycine and N-acetylglycine yielded both protonated monomers with abundances differing by a factor of only 2; this suggests that in protonated peptides, the amide bonds are competitive with the N-terminal amino group as sites of protonation. It is clear that the propensities to proton’ or metal-cation location at particular sites in peptides are influenced by both short- and long-range intraionic interactions. In peptides composed of amino acids of similar cation affinities, it may be postulated that the ion population is heterogeneous with respect to the site of charge, with consequent promotion of multiple low-energy fragmentation routes.     

Strategies for generating peptide radical cations via ion/ion reactions

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon–iodine (C―I) bond are subjected to UVPD with 266‐nm photons, which selectively cleaves the C―I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even‐electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non‐covalent complexes in the electrospray process. Copyright © 2015 John Wiley & Sons, Ltd.     

Formation and Fragmentation of Protonated Molecules after Ionization of Amino Acid and Lactic Acid Clusters by Collision with Ions in the Gas Phase

Collisions between O³⁺ ions and neutral clusters of amino acids (alanine, valine and glycine) as well as lactic acid are performed in the gas phase, in order to investigate the effect of ionizing radiation on these biologically relevant molecular systems. All monomers and dimers are found to be predominantly protonated, and ab initio quantum–chemical calculations on model systems indicate that for amino acids, this is due to proton transfer within the clusters after ionization. For lactic acid, which has a lower proton affinity than amino acids, a significant non‐negligible amount of the radical cation monomer is observed. New fragment‐ion channels observed from clusters, as opposed to isolated molecules, are assigned to the statistical dissociation of protonated molecules formed upon ionization of the clusters. These new dissociation channels exhibit strong delayed fragmentation on the microsecond time scale, especially after multiple ionization.     

Hierarchical assembly of helicate-type dinuclear titanium(IV) complexes

The ligands 4-7-H(2) were used in coordination studies with titanium(IV) and gallium(III) ions to obtain dimeric complexes Li(4)[(4-7)(6)Ti(2)] and Li(6)[(4/5a)(6)Ga(2)]. The X-ray crystal structures of Li(4)[(4)(6)Ti(2)], Li(4)[(5b)(6)Ti(2)], and Li(4)[(7a)(6)Ti(2)] could be obtained. While these complexes are triply lithium-bridged dimers in the solid state, a monomer/dimer equilibrium is observed in solution by NMR spectroscopy and ESI FT-ICR MS. The stability of the dimer is enhanced by high negative charges (Ti(IV) versus Ga(III)) of the monomers, when the carbonyl units are good donors (aldehydes versus ketones and esters), when the solvent does not efficiently solvate the bridging lithium ions (DMSO versus acetone), and when sterical hindrance is minimized (methyl versus primary and secondary carbon substituents). The dimer is thermodynamically favored by enthalpy as well as entropy. ESI FT-ICR mass spectrometry provides detailed insight into the mechanisms with which monomeric triscatecholate complexes as well as single catechol ligands exchange in the dimers. Tandem mass spectrometric experiments in the gas phase show the dimers to decompose either in a symmetric (Ti) or in an unsymmetric (Ga) fashion when collisionally activated. The differences between the Ti and Ga complexes can be attributed to different electronic properties and a charge-controlled reactivity of the ions in the gas phase. The complexes represent an excellent example for hierarchical self-assembly, in which two different noncovalent interactions of well balanced strengths bring together eleven individual components into one well-defined aggregate.