Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.     

Energy-dependent collision-induced dissociation study of buprenorphine and its synthetic precursors

The collision-induced dissociation (CID) of protonated buprenorphine ([M+H](+) ) and four related compounds was studied by electrospray quadrupole/time-of-flight mass spectrometry (ESI-QTOF MS). The fragmentation pathways were investigated by using energy-dependent CID and pseudo-MS(3) (in-source CID combined with tandem mass spectrometry (MS/MS)) methods. The first steps of the fragmentation are the parallel losses of the substituents from the non-aromatic ring moieties. Depending on the applied collision energies, a large number of further fragment ions arising from the cross-ring cleavages of the core-ring structure were observed. Based on the experimental results, a generalized fragmentation scheme was developed for the five buprenorphine derivatives highlighting the differences for the alternatively substituted compounds. The collision-energy-dependent fragmentation profile of buprenorphine is visualized in a two-dimensional plot to aid its fingerprint identification.     

Effect of metal cationization on the low-energy collision-induced dissociation of loganin, epi-loganin and ketologanin studied by electrospray ionization tandem mass spectrometry

The effect of alkali metal and silver cationization on the collision-induced dissociation (CID) of loganin (1), epi-loganin (2) and ketologanin (3) is discussed. Their protonated molecular ions fragment mainly by glycosidic cleavages. The epimeric pairs (1 and 2) show differences in the abundances of the resulting fragment ions. Lithium cationization induces new dissociation pathways such as the retro-Diels-Alder (RDA) fragmentation followed by rearrangement. Unlike the dissociation of protonated molecular ions, the dissociation of lithiated molecules also provides lithiated sugar fragments. The CID of dilithiated molecules is substantially different from that of the monolithiated precursors. RDA reaction appears to be favoured by the presence of the additional lithium atom in the molecule. In addition, other ring cleavages are also induced. The abundances of the various fragment ions are different in the CID spectra of the epimeric pairs. Extensive D labelling and (6)Li labelling experiments confirmed many of the ion structures proposed. The CID spectra of the sodiated ions are generally weaker, although similar to those of the corresponding lithiated species. Higher alkali metal ion (K(+), Rb(+) and Cs(+)) adducts generated only the corresponding metal ions as products of CID. Similar fragmentations were also observed in the CID of the [M + Ag](+) ions of these compounds, the epimeric pairs showing characteristic differences in their CID behaviour. Copyright 2000 John Wiley & Sons, Ltd.     

Comparative dissociation of peptide polyanions by electron impact and photo-induced electron detachment

We compare product-ion mass spectra produced by electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) of multi-deprotonated peptides on a Fourier transform and a linear ion trap mass spectrometer, respectively. Both methods, EDD and EPD, involve the electron emission-induced formation of a radical oxidized species from a multi-deprotonated precursor peptide. Product-ion mass spectra display mainly fragment ions resulting from backbone cleavages of C_α-C bond ruptures yielding a and x ions. Fragment ions originating from N-C_α backbone bond cleavages are also observed, in particular by EPD. Although EDD and EPD methods involve the generation of a charge-reduced radical anion intermediate by electron emission, the product ion abundance distributions are drastically different. Both processes seem to be triggered by the location and the recombination of radicals (both neutral and cation radicals). Therefore, EPD product ions are predominantly formed near tryptophan and histidine residues, whereas in EDD the negative charge solvation sites on the backbone seem to be the most favorable for the nearby bond dissociation.     

Application of positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry to a study of the fragmentation of 6-hydroxyluteolin 7-O-glucoside and 7-O-glucosyl-(1 --> 3)-glucoside

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the structural characterization of 6-hydroxyluteolin 7-O-glucoside and 7-O-glucosyl-(1 --> 3)-glucoside. In-source fragmentation of both glycosides at an increased potential yielded the protonated and deprotonated aglycone, allowing CID spectra to be obtained. The differentiation between quercetin and 6-hydroxyluteolin aglycones was achieved by product ion analysis of the protonated and deprotonated aglycone (m/z 303 and 301), that showed the characteristic product ions (1,3)A at m/z 151 and 153 for quercetin, and m/z 167 and 169 for 6-hydroxyluteolin, consistent with the trihydroxylated A-ring skeleton. In the negative ion mode both glycosides were shown to undergo collision-induced homolytic and heterolytic cleavages of the O-glycosidic bond producing the aglycone radical-anion [Y0-H]-* and Y0(-) product ions. At lower collision energy, various fragmentations involving the glucose moieties were observed with a relatively higher abundance for the monoglucoside compared to the diglucoside. In the latter case both the inner and the terminal glucose residues were involved in the fragmentations, giving useful information on the 1 --> 3 interglycosidic linkage. CID MS/MS analysis of the sodiated molecules gave complementary information for the structural characterization of the studied compounds. Fragmentation mechanisms are proposed for the observed product ions.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Study of structural characteristic features of phenanthriondolizidine alkaloids by fast atom bombardment with tandem mass spectrometry

The mass spectrometric behavior of phenanthroindolizidine alkaloids has been investigated in detail by positive ion fast atom bombardment tandem mass spectrometry (FAB-MS/MS) combined with collision-induced dissociation (CID). The fragmentation has been correlated with the skeleton structure and positions of substituents. The product ions arising from retro-Diels-Alder cleavages differ clearly for compounds with different skeletons. The substituents at C-14 were observed to markedly influence the fragmentation pathway of [M + H](+) ions. A diversity of dissociation behavior initiated by the variation of substituents on the aromatic ring was also observed in the CID spectra. Product ions resulting from loss of CH(4) were obtained for compounds in which both C-6 and C-7 are occupied by methoxy groups. FAB-MS/MS spectra can reflect the connection between the fragmentation behavior and structural features of these phenanthroindolizidine alkaloids rapidly and effectively, and should prove to be a powerful method to determine the structures of related compounds.     

Study of the dissociation of a charge-reduced phosphopeptide formed by electron transfer from an alkali metal target

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.     

Atmospheric pressure thermal dissociation of phospho- and sulfopeptides

Several phospho- and sulfopeptides were subjected to atmospheric pressure thermal dissociation (APTD), which was effected by passing peptide ions generated by electrosonic spray ionization (ESSI) through a heated coiled metal tube. Sequence informative fragment ions including a-, b-, c-, and y-types of ions were observed with increased relative intensities under APTD compared with collision-induced dissociation (CID), performed inside the ion trap. A certain degree of preservation of phosphate and sulfate ester moieties was observed for some fragments ions under APTD. The neutral fragments generated outside the mass spectrometer were further analyzed via on-line corona discharge to provide rich and complementary sequence information to that provided by the fragment ions directly obtained from APTD, although complete losses of the modification groups were noted. Improved primary sequence information for phospho- and sulfopeptides was typically obtained by analyzing both ionic and neutral fragments from APTD compared with fragment ions from CID alone. Localization of the modification sites of phospho- and sulfopeptides was achieved by combining the structural information acquired from APTD and CID.     

Electron transfer dissociation of N-glycopeptides: loss of the entire N-glycosylated asparagine side chain

The recently introduced electron transfer dissociation (ETD) technique opens new possibilities for the structural characterization of glycoproteins at the glycopeptide level. In this report, we investigate the ETD mass spectra of tryptic N-glycopeptides of the model glycoprotein horseradish peroxidase (HRP). Multiply protonated N-glycopeptides obtained by electrospray ionization were subjected to ETD. Fragment ions obtained by ETD were further analyzed by collision-induced dissociation (CID) (MS(3)) for their unambiguous structural assignment. The following fragmentation features were revealed: (1) c- and z-type peptide backbone cleavages were observed with retention of the intact glycan moiety revealing peptide sequence, glycan attachment site, and glycan mass; (2) to a lesser extent, glycosidic bond cleavages were registered with retention of the intact peptide sequence; and (3) a range of amino acid side chain losses did occur. Remarkably, the loss of the complete N-glycosylated asparagine side chain was observed. This loss of the glycan-modified side chain helps with the structural characterization of glycopeptides by allowing the facile deduction and verification of the glycan mass and the nature of the amino acid residue at the glycan attachment site. Importantly, informative ETD spectra were obtained in this study by reversed-phase nano-liquid chromatography (LC) coupled online to a radio-frequency (rf) quadrupole ion trap (QIT) mass spectrometer with alternating acquisition of CID and ETD mass spectra from an automatically selected set of precursors (data-dependent mode). Thus, our study brings nano-LC/QIT-MS(n) with CID and ETD to the fore as a powerful technique for glycoproteomics at the glycopeptide level.     

Tandem mass spectrometry of Cu(II) complexes: the effects of ligand donor group on dissociation

A quadrupole ion trap mass spectrometer was used to study the dissociation patterns of Cu(II) complexes with various linear podand ligands. Cu(II) complexes having different combinations of nitrogen-, oxygen- and sulfur-containing terminal functionality attached to a diethylenetriamine (DIEN) framework were ionized by electrospray and collision-induced dissociation (CID) was used to generate product ions. Regardless of the particular functional groups present, the complexes undergo predominantly heterolytic cleavages of carbon-carbon bonds along the DIEN backbone with Cu remaining coordinated to one of the two terminal functional groups. Upon dissociation, Cu's preference to remain coordinated to a particular functional group follows the trend thioether > amine > imidazole > pyridine > ether. A simple evaluation of this trend based upon metal-functional group binding affinity appears not to be adequate for fully explaining these observations. The tendency of Cu(II) to be reduced upon dissociation helps explain the observed trend, as does the flexibility of the functional group, which affects its ability to orient its dipole effectively toward the metal.     

N,N′-双-(2-羟基苯亚甲基)-烷基(芳基)甲二胺类化合物质谱研究

本文对双Schiff碱类化合物的质谱进行了研究,由于其结构的特殊性,质谱的裂解过程中出现一些特殊的碎片离子,它包括羟基氢的迁移、双键的转换以及分子结构重排等,为研究这类化合物的结构和性质提供了重要的信息。     

High energy collision-induced dissociation of alkali-metal ion adducts of crown ethers and acyclic analogs.

High energy collision-induced dissociation (CID) techniques were applied for structural elucidation of alkali-metal ion adducts of crown ethers. The CID of alkali-metal adducts of tetraglyme and hexaethylene glycol were also evaluated to contrast the fragmentation pathways of the cyclic ethers with those of acyclic analogs. A common fragmentation channel for alkali-metal ion adducts of all the ethers, which results in distonic radical cations, is the homolytic cleavage of carbon-carbon bonds. Additionally, dissociation by carbon-oxygen bond cleavages occurs, and these processes are analogous to the fragmentation pathways observed for simple protonated ethers. The proposed fragmentation pathways for alkali-metal ion adducts of crown ethers result mostly in odd-electron, acyclic product ions. Dissociation of the alkali-metal ion adducts of the acyclic ethers is dominated by losses of various neutral species after an initial hydride or proton transfer. The CID processes for all ethers are independent of the alkali-metal ion sizes; however, the extent of dissociation of the complexes to bare alkali-metal ions increases with the size of the metal.     

A novel tandem quadrupole mass spectrometer allowing gaseous collisional activation and surface induced dissociation.

A novel tandem quadrupole mass spectrometer is described that enables gaseous collision-induced dissociation (CID) and surface-induced dissociation (SID) experiments. The instrument consists of a commercially available triple quadrupole mass spectrometer connected to an SID region and an additional, orthogonal quadrupole mass analyser. The performance of the instrument was evaluated using leucine-enkephalin, allowing a comparison between CID and SID, and with previous reports of other SID instruments. The reproducibility of SID data was assessed by replicate determinations of the collision energy required for 50% dissociation of leucine-enkephalin; excellent precision was observed (standard deviation of 0.6 eV) though, unexpectedly, the reproducibility of the equivalent figure for CID was superior. Several peptides were analysed using SID in conjunction with liquid secondary-ion mass spectrometry or electrospray; a comparison of the fragmentation of singly protonated peptide ions and the further dissociation of y-type fragments was consistent with the equivalence of the latter fragments to protonated peptides. Few product ions attributable to high-energy cleavages of amino acid side-chains were observed. The SID properties were investigated of a series of peptides differing only in the derivatization of a cysteine residue; similar decomposition efficiencies were observed for all except the cysteic acid analogue, which demonstrated significantly more facile fragmentation.     

Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization

We applied electrospray ionization (ESI) tandem quadrupole mass spectrometry to establish the fragmentation pathways of ceramides under low energy collisional-activated dissociation (CAD) by studying more than thirty compounds in nine subclasses. The product-ion spectra of the [M + Li]+ ions of ceramides contain abundant fragment ions that identify the fatty acyl substituent and the long-chain base (LCB) of the molecules, and thus, the structure of ceramides can be easily determined. Fragment ions specific to each ceramide subclasses are also observed. These feature ions permit differentiation among different ceramide subclasses. The ion series arising from the classical C-C bond cleavages that were reported in the fast-atom bombardment (FAB)-high energy tandem mass spectrometry is not observable; however, the product-ion spectra contain multiple fragment ions informative for structural characterization and isomer identification. We also investigated the tandem mass spectra of the fragment ions generated by in-source CAD (pseudo-MS3) and of the deuterium-labeling molecular species obtained by H/D exchange to support the ion structure assignments and the proposed fragmentation pathways that lead to the ion formation.     

Comparison of collision-induced dissociation and electron-induced dissociation of <Emphasis Type="Italic">singly protonated</Emphasis> aromatic amino acids,cystine and related simple peptides using a hybrid linear ion trap–FT-ICR mass spectrometer

The gas-phase fragmentation reactions of singly protonated aromatic amino acids, their simple peptides as well as simple models for intermolecular disulfide bonds have been examined using a commercially available hybrid linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Low-energy collision-induced dissociation (CID) reactions within the linear ion trap are compared with electron-induced dissociation (EID) reactions within the FT-ICR cell. Dramatic differences are observed between low-energy CID (which occurs via vibrational excitation) and EID. For example, the aromatic amino acids mainly fragment via competitive losses of NH(3) and (H(2)O+CO) under CID conditions, while side-chain benzyl cations are major fragment ions under EID conditions. EID also appears to be superior in cleaving the S-S and S-C bonds of models of peptides containing an intermolecular disulfide bond. Systematic studies involving fragmentation as a function of electron energy reveal that the fragmentation efficiency for EID occurs at high electron energy (more than 10 eV) compared with the low-electron energy (less than 0.2 eV) typically observed for electron capture dissociation fragmentation. Finally, owing to similarities between the types of fragment ions observed under EID conditions and those previously reported in ultraviolet photodissociation experiments and the electron-ionization mass spectra, we propose that EID results in fragmentation via electronic excitation and vibrational excitation. EID may find applications in analyzing singly charged molecular ions formed by matrix-assisted laser desorption ionization.     

Gas Phase Chemistry of Li+ with Amides: the Observation of LiOH Loss in Mass Spectrometry

Collision-induced dissociation (CID) of Li(+) adducts of three sets of compounds that contains an amide bond, including 2-(4, 6-dimethoxypyrimidin-2-ylsulfanyl)-N-phenylbenzamide, its derivatives and simpler structures was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Observed fragment ions include those that reflect loss of LiOH. Other product ions result from the Smiles rearrangement and direct C-S bond cleavage. MS/MS of H/D exchange products demonstrated occurrence of a 1,3-H shift from the amide nitrogen atom to the phenyl ring of these compounds. The LiOH loss from Li(+) adducts of amides was further examined by CID of [M + Li](+) ions of N-phenylbenzamide and N-phenylcinnamide. Loss of LiOH was essentially the sole fragmentation reaction observed for the former. For the latter, both losses of LiOH and H(2)O were discovered. The presence of electron-donating substituents of the phenyl ring of these compounds was found to facilitate elimination of LiOH, while that loss was retarded by electron-withdrawing substituents. Proposed fragment ion structures were supported by elemental compositions deduced from ultrahigh resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR-MS/MS) m/z value determinations. Density functional theory-based (DFT) calculations were performed to evaluate potential mechanisms for these reactions.     

Increased sequence coverage of thymine‐rich oligodeoxynucleotides by infrared multiphoton dissociation compared to collision‐induced dissociation

Infrared multiphoton dissociation (IRMPD) of thymine‐rich oligodeoxynucleotides in a linear ion‐trap mass spectrometer affords far more extensive fragmentation than conventional collision‐induced dissociation (CID). For oligodeoxynucleotides containing one non‐thymine base, CID results primarily in cleavage on the 3′ side of the non‐thymine nucleobase, whereas IRMPD results in cleavages between all the nucleobases and thus provides complete sequence coverage. Furthermore, for oligodeoxynucleotides containing a single non‐thymine base, it is shown that the full series of diagnostic sequence ions observed in the IRMPD mass spectra arise from secondary dissociation of the two primary products formed from the initial cleavage site located next to the non‐thymine base. Copyright © 2010 John Wiley & Sons, Ltd.     

High-energy collision-induced dissociation of ceramide ions from permethylated glycosphingolipids

Ceramide fragments from permethylated glycosphingolipids (GSLs) were studied by highenergy collision-induced dissociation (CID). In comparison with ceramlde fragments of underivatized GSLs, many more product ions including charge-remote fragment ions were observed. These ions provided detailed structural information on the ceramides. The relative intensity and the mass interval between the L and M ions were used to assign the position of the double bond. The position of the hydroxyl group was assigned with the Ln and K ions. Because the ceramide fragments and not the pseudomolecular ions were selected as the precursor ions, the size of GSLs had little effect on the quality of the product ion spectra. The sensitivity of this approach was in the range of picomoles.     

Determination of collision-induced dissociation mechanisms and cross-sections in organophosphorus compounds by atmospheric pressure ionization tandem mass spectrometry

Nineteen organophosphorus compounds (OPCs) of the generic classes (dialkyl alkyl′-phosphonates (twelve compounds) and tri-alkyl phosphates (seven compounds) were investigated by atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). All OPCs showed consistent and comparable collision-induced dissociation (CID) pathways allowing for a generalized scheme for dissociation. When the alkyl groups are equal to or larger than ethyl, CID mechanisms are dominated by McLafferty rearrangements and neutral losses are stable molecules in all cases except cleavage of P? C or O? C bonds to form alkyl fragment ions. CID cross-sections for the OPCs were determined with excellent reproducibility and a good correlation was found with a simple model. Inferences with respect to ionic structures were made from observed trends in the cross-section measurements. Limitations in API-MS/MS instruments of this design are found in the energy distribution of the ionization and ion sampling events.