Halogeno-substituted 2-aminobenzoic acid derivatives for negative ion fragmentation studies of N-linked carbohydrates

Negative ion electrospray mass spectra of high-mannose N-linked glycans derivatised with 2-aminobenzoic acids and ionised from solutions containing ammonium hydroxide gave prominent [M-H](-) ions accompanied by weaker [M-2H](2-) ions. Fragmentation of both types of ions gave prominent singly charged glycosidic cleavage ions containing the derivatised reducing terminus and ions from the non-reducing terminus that appeared to be products of cross-ring cleavages. Differentiation of these two groups of ions was conveniently achieved in a single spectrum by use of chloro- or bromo-substituted benzoic acids in order to label ions containing the derivative with an atom with a distinctive isotope pattern. Fragmentation of the doubly charged ions gave more abundant fragments, both singly and doubly charged, than did fragmentation of the singly charged ions, but information of chain branching was masked by the appearance of prominent ions produced by internal cleavages.     

Collision-induced fragmentation of negative ions from N-linked glycans derivatized with 2-aminobenzoic acid

N-Linked glycans from bovine ribonuclease B, chicken ovalbumin, bovine fetuin, porcine thyroglobulin and human alpha(1)-acid glycoprotein were derivatized with 2-aminobenzoic acid by reductive amination and their tandem mass spectra were recorded by negative ion electrospray ionization with a quadrupole time-of-flight mass spectrometer. Derivatives were also prepared from 2-amino-5-methyl- and 2-amino-4,5-dimethoxybenzoic acid in order to confirm the identity of fragment ions containing the reducing terminus. Major fragments from the [M - H](-) ions from the neutral glycans retained the derivative (Y-type cleavages) and provided information on sequence and branching. Other major fragments were products of A-type cross-ring cleavages giving information on antenna structure. Singly doubly and triply charged ions were formed from sialylated glycans. They produced major fragments by loss of sialic acid and a series of singly charged ions that were similar to those from the neutral analogues. Doubly charge ions were also produced by the neutral glycans and were fragmented to form product ions with one and two charges. Again, the fragment ions with a single charge were similar to those from the singly charged parents, but branching information was less obvious because of the occurrence of more abundant ions produced by multiple cleavages. Detection limits were around 200 fmol (3 : 1 signal-to-noise ratio).     

Multiple lithium exchange under lithium cationization of cyclodextrins

Multiple lithium exchange is observed during electrospray ionization of alpha-, beta- and gamma-cyclodextrins from aqueous methanolic solution containing LiOH. Apart from [M + Li](+) and [M + nLi - (n - 1)H](+) ions, abundant multiply lithiated doubly charged ions corresponding to [M + nLi - (n - 2)H](2+) ions were observed. At least six lithium exchanges in alpha-cyclodextrin, seven in beta-cyclodextrin and eight in gamma-cyclodextrin were noted. The propensity of multiply lithiated doubly charged ions is much less in the open-ended maltoheptaose. It appears that during droplet or cluster formation and subsequent desolvation, LiOH trapped in the cavity of cyclodextrin reacts to form multiply lithiated ions. The singly charged [M + Li](+) and doubly charged [M + 2Li](2+) ions fragment by glycosidic cleavages, giving B series of ions, whereas the multiply lithiated ions fragment by cross ring cleavages ((2, 4)A or (O, 2)X) followed by glycosidic cleavage. From the tandem mass spectra, it appears that a maximum of two lithium exchanges occur in one sugar unit in these cyclodextrins.     

Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus

Derivatives were prepared from N-linked glycans by reductive amination from 2-aminobenzamide, 2-aminopyridine, 3-aminoquinoline, 2-aminoacridone, 4-amino-N-(2-diethylaminoethyl)benzamide, and the methyl, ethyl, and butyl esters of 4-aminobenzoic acid. Their electrospray and collision-induced dissociation (CID) fragmentation spectra were examined with a Q-TOF mass spectrometer. The strongest signals were obtained from the [M + Na]+ ions for all derivatives except sugars derivatized with 4-amino-N-(2-diethylaminoethyl)benzamide which gave very strong doubly charged [M + H + Na]2+ ions. The strongest [M + Na]+ ion signals were obtained from the butyl ester of 4-aminobenzoic acid and the weakest from 2-aminopyridine. The most informative spectra were recorded from the [M + Li]+ or [M + Na]+ ions. These spectra were dominated by ions produced by sequence-revealing glycosidic cleavages and "internal" fragments. Linkage-revealing cross-ring cleavage ions were reasonably abundant, particularly from high-mannose glycans. Although the nature of the derivative was found to have little effect upon the fragmentation pattern, 3-aminoquinoline derivatives gave marginally more abundant cross-ring fragments than the other derivatives. [M + H]+ ions formed only glycosidic fragments with few, if any, cross-ring cleavage ions. Doubly charged molecular ions gave less informative spectra; singly charged fragments were weak, and molecular ions containing hydrogen ([M + 2H]2+ and [M + H + Na]2+) fragmented as the [M + H]+ singly charged ions with no significant cross-ring cleavages.     

Collision-induced fragmentation of underivatized N-linked carbohydrates ionized by electrospray

The electrospray mass spectra and collision-induced fragmentation of neutral N-linked glycans obtained from glycoproteins were examined with a Q-TOF mass spectrometer. The glycans were ionized most effectively as adducts of alkali metals, with lithium providing the most abundant signal and caesium the least. Singly charged ions generally gave higher ion currents than doubly charged ions. Addition of formic acid could be used to produce [M + H]+ ions, but these ions were always accompanied by abundant cone-voltage fragments. The energy required for collision-induced fragmentation was found to increase in a linear manner as a function of mass with the [M + Na]+ ions requiring about four times as much energy as the [M + H]+ ions for complete fragmentation of the molecular ions. Fragmentation of the [M + H]+ ions gave predominantly B- and Y-type glycosidic fragments whereas the [M + Na]+ and [M + Li]+ ions produced a number of additional fragments including those derived from cross-ring cleavages. Little fragmentation was observed from the [M + K]+ and [M + Rb]+ ions and the only fragment to be observed from the [M + Cs]+ ion was Cs+. The [M + Na]+ and [M + Li]+ ions from all the N-linked glycans gave abundant fragments resulting from loss of the terminal GlcNAc moiety and prominent, though weaker, ions as the result of 0,2A and 2,4A cross-ring cleavages of this residue. Most other ions were the result of successive additional losses of residues from the non-reducing terminus. This pattern was particularly prominent with glycans containing several non-reducing GlcNAc residues where successive losses of 203 u were observed. Many of the ions in the low-mass range were products of several different fragmentation routes but still provided structural information. Possibly of most diagnostic importance was an ion formed by loss of 221 u (GlcNAc molecule) from an ion that had lost the 3-antenna and the chitobiose core. This latter ion, although coincident in mass with some other 'internal' fragments, often provided additional information on the composition of the antennae. Other ions defining antenna composition were weak cross-ring fragments produced from the core branching mannose residue. Glycans containing Gal-GlcNAc residues showed successive losses of this moiety, particularly from the B-type fragments resulting from loss of the reducing-terminal GlcNAc residue. The [M + Na]+ and [M + Li]+ ions from high-mannose and hybrid glycans gave a series of ions of composition (Man)nNa/Li+ where n = 1 to the total number of glycans in the molecule, allowing these sugars to be distinguished from the more highly processed complex glycans. Other ions in the spectra of the high-mannose glycans were diagnostic of chain branching but insufficient information was available to determine their mode of formation.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

Elucidation of high micro-heterogeneity of an acidic-neutral trichotoxin mixture from Trichoderma harzianum by electrospray ionization quadrupole time-of-flight mass spectrometry

The high micro-heterogeneity of an acidic-neutral trichotoxin mixture from T. harzianum, PC01, was elucidated using a modern tandem mass spectrometer equipped with an electrospray ionization source, a hybrid quadrupole-orthogonal accelerator and a reflectron time-of-flight analyzer. The trichotoxins appeared predominantly in six possible doubly charged pseudo molecular ions with three different adducts (H, Na and K) as [M + 2H](2+), [M + H + Na](2+), [M + H + K](2+), [M + 2Na](2+), [M + Na + K](2+) and [M + 2K](2+). The singly charged pseudomolecular ions, [M + H](+), [M + Na](+) and [M + K](+), occurred only in low abundance when the cone voltages were higher than 30 V. Additional singly charged fragments, b(12) and y"6 (complementary N- and C-terminal fragments), were obtained in high abundance using high cone voltages. The peak patterns of both singly and doubly charged molecular adducts revealed that this trichotoxin mixture contained several components having 6-7 molecular masses with a consecutive 14 u difference among members in the same molecular adduct series. Furthermore, well resolved isotopic peaks of every doubly or singly charged ions and their reproducible peak intensity allowed the identification of the mixing of acidic trichotoxins 1 u molecular mass heavier than the neutral counterparts in the sample. Tandem mass spectrometric (MS/MS) analyses of various singly charged b(12) and y"6 ions supported the sequence deduction of the major and minor components and also the position of Glu in the sequences of these acidic molecules. The setting of either low or high resolution of the quadrupole mass filter unit together with a suitable variation of the collision voltage for any MS/MS precursor were the tools for extracting a number of mixed components and obtaining the major and minor sequences of these precursor peaks. The nature of the MS/MS fragmentation and the data assignment of three major doubly charged ions, [M + 2H](2+), [M + K + H](2+) and [M + 2K](2+), are demonstrated. Eleven new sequences of both acidic and neutral trichotoxins are reported.     

Ionization and collision-induced fragmentation of N-linked and related carbohydrates using divalent cations

Maltoheptaose and several N-linked glycans were ionized by electrospray as adducts with the divalent cations Mg2+, Ca2+, Mn2+, Co2+ and Cu2+. [M + metal]2+ ions were the major species in all cases with calcium giving the highest sensitivity. In addition, copper gave [M + Cu]+ ions. Other cations gave singly charged ions only by elimination of a protonated monosaccharide. Fragmentation of the [M + metal]2+ ions produced both singly and doubly charged ions with the relative abundance of doubly charged ions decreasing in the order Ca > Mg > Mn > Co > Cu. Singly charged ions were formed by elimination of a protonated monosaccharide residue followed, either by successive monosaccharide residue losses, or by a 2,4A cross-ring cleavage of the reducing-terminal monosaccharide. Formation of doubly charged fragments from [M + metal]2+ ions involved successive monosaccharide-residue losses either with or without O,2A or 2,4A cross-ring cleavages of the reducing-terminal monosaccharide. Abundant diagnostic doubly charged ions formed by loss of the 3-antenna from the O,2A cross-ring product were specific to [M + Ca]2+ ions. Fragmentation of [M + Cu]+ ions was similar to that of the corresponding [M + H]+ ions in that most cross-ring fragments were absent.     

Ionization and fragmentation of N-linked glycans as silver adducts by electrospray mass spectrometry

[M+Ag]+ ions were produced by electrospray from neutral high-mannose, hybrid and complex N-linked glycans obtained from bovine ribonuclease, chicken egg glycoproteins, bovine fetuin and porcine thyroglobulin by the addition of silver nitrate to the electrospray solvent. Both singly and doubly charged ions were produced but, as the signals were split between the two silver isotopes, sensitivity was not as high as with the sodium adducts reported earlier. Collision-induced dissociation (CID) spectra were dominated by ions produced by glycosidic cleavages, mainly of the B- and Y-type. Internal cleavage ions involving both B and Y cleavages were very prominent but cross-ring fragments were generally of very low abundance or absent. Silver was very efficient at cleaving the glycosidic bonds, so much so that spectra tended to contain glycosidic ions at most possible combinations of the constituent monosaccharides.     

Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides.

The presence of disulfide linkages in multiply charged polypeptide ions tends to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions. In particular, fragmentation that requires two cleavages (i.e., cleavage of a disulfide linkage and a peptide linkage) is strongly suppressed. Reduction of the disulfide linkage(s) by use of dithiothreitol yields parent ions upon electrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced species than from the native species. These observations are illustrated with doubly protonated native and reduced somatosin, the [M + 5H](5+) ion of native bovine insulin and the [M + 4H](4+) and [M + 3H](3+) ions of the B-chain of bovine insulin produced by reduction of the disulfide linkages in insulin, and the [M + 11H](11+) ion of native chicken lysozyme and the [M + 11H](11+) and [M + 14H](14+) ions of reduced lysozyme. In each case, the product ions produced by ion trap collisional activation were subjected to ion/ion proton transfer reactions to facilitate interpretation of the product ion spectra. These studies clearly suggest that the identification of polypeptides with one or more disulfide linkages via application of ion trap collisional activation to the multiply charged parent ions formed directly by electrospray could be problematic. Means for cleaving the disulfide linkage, such as reduction by dithiothreitol prior to electrospray, are therefore desirable in these cases.     

Collisionally activated dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides

Doubly charged sodiated and permethylated linear malto-oligosaccharides ({Glc}6-{Glc}9), branched N-linked glycans (high-mannose type GlcNAc2Man5-9, and complex asialo- and disialylated-biantennary glycans) were analyzed by tandem mass spectrometry using collisionally-activated dissociation (CAD) and "hot" electron capture dissociation (ECD) available in a custom-built ESI FTICR mass spectrometer. For linear permethylated malto-oligosaccharides, both CAD and "hot" ECD produced glycosidic cleavages (B, Y, C, and Z ions), cross-ring cleavages (A- and X-type), and internal cleavages (B/Y and C/Y type) to provide sequence and linkage information. For the branched N-linked glycans, CAD and "hot" ECD provided complementary structural information. CAD generated abundant B and Y fragment ions by glycosidic cleavages, whereas "hot" ECD produced dominant C and Z ions. A-type cross-ring cleavages were present in CAD spectra. Complementary A- and X-type cross-ring fragmentation pairs were generated by "hot" ECD, and these delineated the branching patterns and linkage positions. For example, 0, 4An and 3, 5An ions defined the linkage position of the major branch as the 6-position of the central core mannose residue. The internal fragments observed in CAD were more numerous and abundant than in "hot" ECD spectra. Since the triply charged (sodiated) molecular ion of the permethylated disialylated-biantennary N-linked glycan has relatively high abundance, it was isolated and fragmented in a "hot" ECD experiment and extensive fragment ions (glycosidic and complementary pairs of cross-ring cleavages) were generated to fully confirm the sequence, branching, and linkage assignments for this glycan.     

Characterization of peptides by liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a post-column complexant

Two model peptides, des-Arg1-bradykinin (DAB) and bradykinin (B), were cationized by Ag+ after their separation by reversed-phase liquid chromatography (RPLC) prior to mass spectrometry (MS). Silver nitrate solution was used as a post-column reagent. The RPLC and MS experimental conditions were optimized using flow injection in order to obtain sufficiently abundant silver adducts to permit MS/MS experiments. The use of water-methanol with 0.1% formic acid as mobile phase allowed a good chromatographic separation of the two peptides with a polymeric stationary phase and sufficiently abundant silver-containing adducts, [M + Ag + H]2+ and [M + 2Ag]2+. The gas-phase dissociation of [DAB + Ag + H]2+ and [DAB + 2Ag]2+ led to interpretable mass spectra during the on-line cationization experiment. Most of the ions obtained by dissociating [DAB + Ag + H]2+ and [DAB + 2Ag]2+ species are silver-containing ions but the ions produced depend on the parent. The ions coming from the dissociation of the doubly charged silver adducts [DAB + Ag + H]2+ or [DAB + 2Ag]2+ are of interest compared with those coming from the singly charged silver species or doubly charged protonated species. The fragmentation of the doubly charged silver adducts provides ions over the entire mass range. Although the presence of several prolines in des-Arg1-bradykinin prevents the formation of some expected ions, the observation of triplets [an-H + Ag]+, [bn-H + Ag]+ and [bn + OH + Ag]+ produced by the dissociation of on-line Ag(+)-cationized peptides could contribute to greater success of automatic sequencing of peptides.     

Non-covalent interactions of alkali metal cations with singly charged tryptic peptides

The complexes formed by alkali metal cations (Cat(+) = Li(+), Na(+), K(+), Rb(+)) and singly charged tryptic peptides were investigated by combining results from the low-energy collision-induced dissociation (CID) and ion mobility experiments with molecular dynamics and density functional theory calculations. The structure and reactivity of [M + H + Cat](2+) tryptic peptides is greatly influenced by charge repulsion as well as the ability of the peptide to solvate charge points. Charge separation between fragment ions occurs upon dissociation, i.e. b ions tend to be alkali metal cationised while y ions are protonated, suggesting the location of the cation towards the peptide N-terminus. The low-energy dissociation channels were found to be strongly dependant on the cation size. Complexes containing smaller cations (Li(+) or Na(+)) dissociate predominantly by sequence-specific cleavages, whereas the main process for complexes containing larger cations (Rb(+)) is cation expulsion and formation of [M + H](+). The obtained structural data might suggest a relationship between the peptide primary structure and the nature of the cation coordination shell. Peptides with a significant number of side chain carbonyl oxygens provide good charge solvation without the need for involving peptide bond carbonyl groups and thus forming a tight globular structure. However, due to the lack of the conformational flexibility which would allow effective solvation of both charges (the cation and the proton) peptides with seven or less amino acids are unable to form sufficiently abundant [M + H + Cat](2+) ion. Finally, the fact that [M + H + Cat](2+) peptides dissociate similarly as [M + H](+) (via sequence-specific cleavages, however, with the additional formation of alkali metal cationised b ions) offers a way for generating the low-energy CID spectra of 'singly charged' tryptic peptides.     

Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry

The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of [M + H]+, [M + Na]+, [M - H + 2Na]+ ions in the positive mode and [M - H]-, [M + Na - 2H]- ions in the negative mode. The AB and PHN derivatives formed abundant [M + H]+ and [M - H]- ions in ESI, and by matrix-assisted laser desorption/ionization (MALDI) produced abundant [M + Na]+ ions. PMP- and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M + Na]+ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Here, PMP produced dominant Y-cleavages from the chitobiose while other ions produced weak signals.     

Electron capture in charge-tagged peptides. Evidence for the role of excited electronic states

Electron capture dissociation (ECD) was studied with doubly charged dipeptide ions that were tagged with fixed-charge tris-(2,4,6-trimethoxyphenyl)phosphonium-methylenecarboxamido (TMPP-ac) groups. Dipeptides GK, KG, AK, KA, and GR were each selectively tagged with one TMPP-ac group at the N-terminal amino group while the other charge was introduced by protonation at the lysine or arginine side-chain groups to give (TMPP-ac-peptide + H)(2+) ions by electrospray ionization. Doubly tagged peptide derivatives were also prepared from GK, KG, AK, and KA in which the fixed-charge TMPP-ac groups were attached to the N-terminal and lysine side-chain amino groups to give (TMPP-ac-peptide-ac-TMPP)(2+) dications by electrospray. ECD of (TMPP-ac-peptide + H)(2+) resulted in 72% to 84% conversion to singly charged dissociation products while no intact charge-reduced (TMPP-ac-dipeptide + H)(+) ions were detected. The dissociations involved loss of H, formation of (TMPP + H)(+), and N-C(alpha) bond cleavages giving TMPP-CH(2)CONH(2)(+) (c(0)) and c(1) fragments. In contrast, ECD of (TMPP-ac-peptide-ac-TMPP)(2+) resulted in 31% to 40% conversion to dissociation products due to loss of neutral TMPP molecules and 2,4,6-trimethoxyphenyl radicals. No peptide backbone cleavages were observed for the doubly tagged peptide ions. Ab initio and density functional theory calculations for (Ph(3)P-ac-GK + H)(2+) and (H(3)P-ac-GK + H)(2+) analogs indicated that the doubly charged ions contained the lysine side-chain NH(3)(+) group internally solvated by the COOH group. The distance between the charge-carrying phosphonium and ammonium atoms was calculated to be 13.1-13.2 A in the most stable dication conformers. The intrinsic recombination energies of the TMPP(+)-ac and (GK + H)(+) moieties, 2.7 and 3.15 eV, respectively, indicated that upon electron capture the ground electronic states of the (TMPP-ac-peptide + H)(+*) ions retained the charge in the TMPP group. Ground electronic state (TMPP-ac-GK + H)(+*) ions were calculated to spontaneously isomerize by lysine H-atom transfer to the COOH group to form dihydroxycarbinyl radical intermediates with the retention of the charged TMPP group. These can trigger cleavages of the adjacent N-C(alpha) bonds to give rise to the c(1) fragment ions. However, the calculated transition-state energies for GK and GGK models suggested that the ground-state potential energy surface was not favorable for the formation of the abundant c(0) fragment ions. This pointed to the involvement of excited electronic states according to the Utah-Washington mechanism of ECD.     

Structural determination of the novel fragmentation routes of zwitteronic morphine opiate antagonists naloxonazine and naloxone hydrochlorides using electrospray ionization tandem mass spectrometry

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of the zwitteronic salts naloxonazine dihydrochloride 1 and naloxone hydrochloride 2, a common series of morphine opiate receptor antagonists, were recorded using different declustering potentials. The singly charged ion [M+H-2HCl](+) at m/z 651.3170 and the doubly charged ion [M+2H-2HCl](2+) at m/z 326.1700 were noted for naloxonazine dihydrochloride 1; and the singly charged ion [M+H-HCl](+) at m/z 328.1541 was observed for naloxone hydrochloride 2. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) experiments established the fragmentation routes of these compounds. In addition to the characteristic diagnostic product ions obtained, we noticed the formation of a series of radical product ions for the zwitteronic compounds 1 and 2, and also the formation of a distonic ion product formed from the singly charged ion [M+H-HCl](+) of naloxone hydrochloride 2. Confirmation of the various established fragmentation routes was effected by conducting a series of ESI-CID-QqTof-MS/MS product ion scans, which were initiated by CID in the atmospheric pressure/vacuum interface using a higher declustering potential. Deuterium labeling was also performed on the zwitteronic salts 1 and 2, in which the hydrogen atoms of the OH and NH groups were exchanged with deuterium atoms. Low-energy CID-QqTof-MS/MS product ion scans of the singly charged and doubly charged deuteriated molecules confirmed the initial fragmentation patterns proposed for the protonated molecules. Precursor ion scan analyses were also performed with a conventional quadrupole-hexapole-quadrupole tandem mass spectrometer and allowed the confirmation of the genesis of some diagnostic ions.     

Effects of transition metal ion coordination on the collision-induced dissociation of polyalanines

Transition metal-polyalanine complexes were analyzed in a high-capacity quadrupole ion trap after electrospray ionization. Polyalanines have no polar amino acid side chains to coordinate metal ions, thus allowing the effects metal ion interaction with the peptide backbone to be explored. Positive mode mass spectra produced from peptides mixed with salts of the first row transition metals Cr(III), Fe(II), Fe(III), Co(II), Ni(II), Cu(I), and Cu(II) yield singly and doubly charged metallated ions. These precursor ions undergo collision-induced dissociation (CID) to give almost exclusively metallated N-terminal product ions whose types and relative abundances depend on the identity of the transition metal. For example, Cr(III)-cationized peptides yield CID spectra that are complex and have several neutral losses, whereas Fe(III)-cationized peptides dissociate to give intense non-metallated products. The addition of Cu(II) shows the most promise for sequencing. Spectra obtained from the CID of singly and doubly charged Cu-heptaalanine ions, [M + Cu - H](+) and [M + Cu](2+) , are complimentary and together provide cleavage at every residue and no neutral losses. (This contrasts with [M + H](+) of heptaalanine, where CID does not provide backbone ions to sequence the first three residues.) Transition metal cationization produces abundant metallated a-ions by CID, unlike protonated peptides that produce primarily b- and y-ions. The prominence of metallated a-ions is interesting because they do not always form from b-ions. Tandem mass spectrometry on metallated (Met = metal) a- and b-ions indicate that [b(n) + Met - H](2+) lose CO to form [a(n) + Met - H](2+), mimicking protonated structures. In contrast, [a(n) + Met - H](2+) eliminate an amino acid residue to form [a(n-1) + Met - H](2+), which may be useful in sequencing.     

Collision-induced dissociation tandem mass spectrometry of desferrioxamine siderophore complexes from electrospray ionization of UO2(2+), Fe3+ and Ca2+ solutions

Desferrioxamine (DEF) is a trihydroxamate siderophore typical of those produced by bacteria and fungi for the purpose of scavenging Fe(3+) from environments where the element is in short supply. Since this class of molecules has excellent chelating properties, reaction with metal contaminants such as actinide species can also occur. The complexes that are formed can be mobile in the environment. Because the natural environment is extremely diverse, strategies are needed for the identification of metal complexes in aqueous matrices having a high degree of chemical heterogeneity, and electrospray ionization mass spectrometry (ESI-MS) has been highly effective for the characterization of siderophore-metal complexes. In this study, ESI-MS of solutions containing DEF and either UO(2)(2+), Fe(3+) or Ca(2+) resulted in generation of abundant singly charged ions corresponding to [UO(2)(DEF - H)](+), [Fe(DEF - 2H)](+) and [Ca(DEF - H)](+). In addition, less abundant doubly charged ions were produced. Mass spectrometry/mass spectrometry (MS/MS) studies of collision-induced dissociation (CID) reactions of protonated DEF and metal-DEF complexes were contrasted and rationalized in terms of ligand structure. In all cases, the most abundant fragmentation reactions involved cleavage of the hydroxamate moieties, consistent with the idea that they are most actively involved with metal complexation. Singly charged complexes tended to be dominated by cleavage of a single hydroxamate, while competitive fragmentation between two hydroxamate moieties increased when the doubly charged complexes were considered. Rupture of amide bonds was also observed, but these were in general less significant than the hydroxamate fragmentations. Several lower abundance fragmentations were unique to the metal examined: abundant loss of H(2)O occurred only for the singly charged UO(2)(2+) complex. Further, NH(3) was eliminated only from the singly charged Fe(3+) complex; this and fragmentation of C-C and C-N bonds derived from neither the hydroxamate nor the amide groups suggested that Fe(3+) insertion reactions were competing with ligand complexation. In no experiments were coordinating solvent molecules observed, attached either to the intact complexes or to the fragment ions, which indicated that both intact DEF and its fragments were occupying all of the coordination sites around the metal centers. This conclusion was based on previous experiments that showed that undercoordinated UO(2)(2+) and Fe(3+) readily added H(2)O and methanol in the ESI quadrupole ion trap mass spectrometer that was used in this study.     

Fragmentation dynamics of methane by few-cycle femtosecond laser pulses

The fragmentation pattern of CH4 was experimentally studied at an intensity of approximately 10(14) W/cm2 with laser durations varying from 8 to 110 fs. When the laser duration was 8 fs, only the primarily fragmental CH3+ ion was observed in addition to the parent CH4+ ion. When the laser duration was 30 fs, small fragmental CH2+ and H+ ions appeared. When the laser duration was 110 fs, some doubly charged ions were also observed in addition to the abundant singly charged ions. The large mass spectra difference demonstrated that the pulse duration had a strong effect on the fragmentation of the parent ion produced in the single ionization. The effect of laser intensity on the fragmentation of CH4+ was also studied for few-cycle femtosecond laser pulses. The results demonstrated that the first-return recollision between the rescattered electron and the parent ion played a significant role in the fragmentation dynamics of the parent ion. Depending on the ion-electron impact energy, the recollision excited the parent ion to a dissociated state or doubly charged state. The experimentally observed singly charged fragmental ions resulted from the recollision-induced dissociation of CH4+ or the Coulomb explosion of CH(4)2+.