Analysis of protonated and alkali metal cationized aminoglycoside antibiotics by collision-activated dissociation and infrared multi-photon dissociation in the quadrupole ion trap

Nine aminoglycoside antibiotics were analyzed in two quadrupole ion trap mass spectrometers using electrospray ionization. Structural information was obtained via collision-activated dissociation (CAD) and infrared multi-photon dissociation (IRMPD) of the protonated species. Several of the compounds, having multiple basic sites, preferred the doubly protonated form while some existed in the singly charged state or were distributed between single and doubly protonated species, allowing comparison of the fragmentation patterns of the two charge states. In general, IRMPD is as efficient as CAD, produces more low-mass fragment ions, and is more universally applied owing to its low dependence on trapping, pressure and tuning conditions. Alkali metal complexation using Li(+) and Na(+) was probed as a means of producing different fragmentation patterns, but in most cases the resulting fragmentation patterns were simplified versions of those obtained for the protonated analogs.     

Electron capture dissociation and infrared multiphoton dissociation of oligodeoxynucleotide dications

We report electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) of doubly protonated and protonated/alkali metal ionized oligodeoxynucleotides. Mass spectra following ECD of the homodeoxynucleotides polydC, polydG, and polydA contain w or d "sequence" ions. For polydC and polydA, the observed fragments are even-electron ions, whereas radical w/d ions are observed for polydG. Base loss is seen for polydG and polydA but is a minor fragmentation pathway in ECD of polydC. We also observe fragment ions corresponding to w/d plus water in the spectra of polydC and d(GCATGC). Although the structure of these ions is not clear, they are suggested to proceed through a pentavalent phosphorane intermediate. The major fragment in ECD of d(GCATGC) is a d ion. Radical a- or z-type fragment ions are observed in most cases. IRMPD primarily results in base loss, but backbone fragmentation is also observed. IRMPD provides more sequence information than ECD, but the spectra are more complex due to extensive base and water losses. It is proposed that the smaller degree of sequence coverage in ECD, with fragmentation mostly occurring close to the ends of the molecules, is a consequence of a mechanism in which the electron is captured at a P=O bond, resulting in a negatively charged phosphate group. Consequently, at least two protons (or alkali metal cations) must be present to observe a w or d fragment ion, a requirement that is less likely for small fragments.     

Studies of rearrangement reactions of protonated and lithium cationized 2-pyrimidinyloxy-N-arylbenzylamine derivatives by MALDI-FT-ICR mass spectrometry

The rearrangement reactions of protonated and lithium-cationized 2-pyrimidinyloxy-N-arylbenzylamine derivatives were studied by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and infrared multiphoton dissociation mass spectrometry (IRMPD). Our results show that three kinds of rearrangement reactions occur in IRMPD processes. First, nearly all protonated 2-pyrimidinyloxy-N-arylbenzylamine derivatives undergo Pathway A to form the K ion series. It is proposed that this rearrangement (migration of a substituted benzyl group) proceeds by way of a gas-phase intramolecular S(N)2 reaction. Second, a gas phase intramolecular S(N)Ar type rearrangement mechanism is proposed to explain the formation of the F ion series from protonated and lithium-cationized 5 (or 6). This skeletal rearrangement reaction competes with the S(N)2 reaction of the Pathway A, which produces the K ion series, in IRMPD of protonated 5 and 6. Third, the formation pathway of the W ion series is explained by a gas phase Cope type rearrangement mechanism.     

Infrared multiphoton dissociation spectroscopic analysis of peptides and oligosaccharides by using fourier transform ion cyclotron resonance mass spectrometry with a midinfrared free-electron laser

The fragmentation of peptides and oligosaccharides in the gas phase was investigated by means of electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry coupled with dissociation by a laser-cleavage infrared multiphoton dissociation (IRMPD) technique. In this technique, an IR free-electron laser is used as a tunable source of IR radiation to cause cleavage of the ionized samples introduced into the FTICR cell. The gas-phase IRMPD spectra of protonated peptides (substance P and angiotensin II) and two sodiated oligosaccharides (sialyl Lewis X and lacto-N-fucopentaose III) were obtained over the IR scan range of 5.7-9.5 microm. In the IRMPD spectra for the peptide, fragment ions are observed as y/b-type fragment ions in the range 5.7-7.5 microm, corresponding to cleavage of the backbone of the parent amino acid sequence, whereas the spectra of the oligosaccharides have major peaks in the range 8.4-9.5 microm, corresponding to photoproducts of the B/Y type.     

Electron detachment dissociation of neutral and sialylated oligosaccharides

Electron detachment dissociation (EDD) has recently been shown by Amster and coworkers to constitute a valuable analytical approach for structural characterization of glycosaminoglycans. Here, we extend the application of EDD to neutral and sialylated oligosaccharides. Both branched and linear structures are examined, to determine whether branching has an effect on EDD fragmentation behavior. EDD spectra are compared to collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) spectra of the doubly and singly deprotonated species. Our results demonstrate that EDD of both neutral and sialylated oligosaccharides provides structural information that is complementary to that obtained from both CAD and IRMPD. In all cases, EDD resulted in additional cross-ring cleavages. In most cases, cross-ring fragmentation obtained by EDD is more extensive than that obtained from IRMPD or CAD. Our results also indicate that branching does not affect EDD fragmentation, contrary to what has been observed for electron capture dissociation (ECD).     

Collision-activated dissociation,infrared multiphoton dissociation,and electron capture dissociation of the <Emphasis Type="Italic">Bacillus anthracis</Emphasis> siderophore petrobactin and its metal ion complexes

Siderophores are high-affinity iron-chelating ligands produced by microorganisms to scavenge vital Fe(3+) from the environment. Thus, siderophores constitute potential therapeutic targets and their structural determination is important for exploiting their therapeutic value. Here, the virulence-associated siderophore petrobactin from Bacillus anthracis was characterized with electron capture dissociation (ECD). Fragmentation of doubly protonated petrobactin was investigated and compared to sustained off-resonance irradiation collision-activated dissociation (SORI CAD) and infrared multiphoton dissociation (IRMPD) of both the singly and doubly protonated species. These experiments demonstrate that ECD provides additional information (complementary bond cleavages) on the structure of petrobactin compared to both SORI CAD and IRMPD. Furthermore, complexes of petrobactin with divalent (Ca(2+), Fe(2+), and Co(2+)) and trivalent (Fe(3+) and Ga(3+)) metal cations were also subjected to SORI CAD and ECD. Again, most structural information was obtained from the ECD spectra. However, significant differences were found in both SORI CAD and ECD of metal complexes, dependent on the nature of the metal ion. Intriguingly, unique behavior, consistent with a recently proposed solution-phase structure, was observed for the highly preferred Fe(3+)-petrobactin complex.     

Structural characterization of the GM1 ganglioside by infrared multiphoton dissociation, electron capture dissociation, and electron detachment dissociation electrospray ionization FT-ICR MS/MS

Gangliosides play important biological roles and structural characterization of both the carbohydrate and the lipid moieties is important. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), electron detachment dissociation (EDD), and infrared multiphoton dissociation (IRMPD) provide extensive fragmentation of the protonated and deprotonated GM1 ganglioside. ECD provides extensive structural information, including identification of both halves of the ceramide and cleavage of the acetyl moiety of the N-acetylated sugars. IRMPD provides similar glycan fragmentation but no cleavage of the acetyl moiety. Cleavage between the fatty acid and the long-chain base of the ceramide moiety is seen in negative-ion IRMPD but not in positive-ion IRMPD of GM1. Furthermore, this extent of fragmentation requires a range of laser powers, whereas all information is available from a single ECD experiment. However, stepwise fragmentation by IRMPD may be used to map the relative labilities for a series of cleavages. EDD provides the alternative of electron-induced fragmentation for negative ions with extensive fragmentation, but suffers from low efficiency as well as complication of data analysis by frequent loss of hydrogen atoms. We also show that analysis of MS/MS data for glycolipids is greatly simplified by classification of product ion masses to specific regions of the ganglioside based solely on mass defect graphical analysis.     

Photoinduced dissociation of heparin-derived oligosaccharides controlled by charge location

The development of strategies based on mass spectrometry to help for deep structural analysis of acidic oligosaccharides remains topical. We thus examined the dissociation behavior of deprotonated ions of heparin-derived di- to tetra-saccharides under UV irradiation at 220 nm. Depending on the ionization state of the carboxylic groups, an oxidized species issued from electron photodetachment was observed in complement to photoinduced fragmentation of precursor ions. The influence of the charge location in the oligosaccharide dianions on the balance between photodissociation and electron photodetachment is examined and a way to direct the relaxation pathways, (i.e., dissociation versus electron detachment), is proposed using sodium adducts. The oxidized species was subjected to activated-electron photodetachment (activated-EPD) leading to complementary informative fragment ions to those issued from photodissociation. Directed photoinduced dissociation at 220 nm and activated-EPD should complement the more conventional CAD and IRMPD activation modes for deeper structural analysis of acidic oligosaccharides-derived anions.     

Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociationof peptides in a quadrupole ion trapwith selective IRMPD of phosphopeptides

Dissociation of protonated peptides via infrared multiphoton dissociation (IRMPD) provides more extensive sequence information than is obtained with collisionally activated dissociation (CAD) in a quadrupole ion trap due to the lack of the CAD low m/z cutoff and the ability to form secondary and higher order fragments with the non-resonant photoactivation technique. In addition, IRMPD is shown to be useful for the selective dissociation of phosphopeptides over those which are not phosphorylated because the greater photon absorption efficiency of the phosphorylated peptides leads to their more rapid dissociation. Finally, the selectivity of the IRMPD technique for phosphorylated species in complex mixtures is confirmed with the analysis of a mock peptide mixture and a tryptic digest of alpha-casein.     

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.     

Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry

An electron injection system based on an indirectly heated ring-shaped dispenser cathode has been developed and installed in a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissociation (ECD) to be carried out by a hollow electron beam coaxial with the ion cyclotron resonance (ICR) trap. Infrared multiphoton dissociation (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the centre of the dispenser cathode. Electron and photon irradiation times of the order of 100 ms are required for efficient ECD and IRMPD, respectively. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, respectively), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR experimental sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MS(n)) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the analysis of protein digests separated by liquid chromatography prior to FTICRMS. Furthermore, ion activation by either electron or laser irradiation prior to, as well as after, dissociation by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.     

Electron capture dissociation of singly and multiply phosphorylated peptides

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.     

An investigation of the energetics of peptide ion dissociation by laser desorption chemical ionization fourier transform mass spectrometry

The energy dependence of competing fragmentation pathways of protonated peptide molecules is studied via laser desorption—chemical ionization in a Fourier transform ion cyclotron resonance spectrometer. Neutral peptide molecules are desorbed by the technique of substrate-assisted laser desorption, followed by post-ionization with a proton transfer reagent ion species. The chemical ionization reaction activates the protonated peptide molecules, which then fragment in accordance with the amount of excess energy that is deposited. Chemical ionization forms a protonated molecule with a narrower distribution of activation energy than can be formed by activation methods such as collision activated dissociation. Furthermore, the upper limit of the activation energy is well defined and is approximately given by the enthalpy of the chemical ionization reaction. Control over the fragmentation of peptide ions is demonstrated through reactions between desorbed peptide molecules with different reagent ion species. The fragmentation behavior of peptide ions with different internal energies is established by generation of a breakdown curve for the peptide under investigation. Breakdown curves are reported for the peptides Val-Pro, Val-Pro-Leu, Phe-Phe-Gly-Leu-Met NH₂, and Arg-Lys-Asp-Val-Tyr. The derived breakdown curve of Val-Pro has been fitted by using quasi-equilibrium Rice-Ramsperger-Kassel-Marcus theory to model the unimolecular dissociation of the protonated peptide to provide a better understanding of the mechanisms for the formation of fragment ions that originate from protonated peptides.     

Infrared multiphoton dissociation of small-interfering RNA anions and cations

Infrared multiphoton dissociation (IRMPD) on a linear ion trap mass spectrometer is applied for the sequencing of small interfering RNA (siRNA). Both single-strand siRNAs and duplex siRNA were characterized by IRMPD, and the results were compared with that obtained by traditional ion trap-based collision induced dissociation (CID). The single-strand siRNA anions were observed to dissociate via cleavage of the 5′ P—O bonds yielding c- and y-type product ions as well as undergo neutral base loss. Full sequence coverage of the siRNA anions was obtained by both IRMPD and CID. While the CID mass spectra were dominated by base loss ions, accounting for ∼25% to 40% of the product ion current, these ions were eliminated through secondary dissociation by increasing the irradiation time in the IRMPD mass spectra to produce higher abundances of informative sequence ions. With longer irradiation times, however, internal ions corresponding to cleavage of two 5′ P—O bonds began to populate the product ion mass spectra as well as higher abundances of [a − Base] and w-type ions. IRMPD of siRNA cations predominantly produced c- and y-type ions with minimal contributions of [a − Base] and w-type ions to the product ion current; the presence of only two complementary series of product ions in the IRMPD mass spectra simplified spectral interpretation. In addition, IRMPD produced high abundances of protonated nucleobases, [G + H]⁺, [A + H]⁺, and [C + H]⁺, which were not detected in the CID mass spectra due to the low-mass cut-off associated with conventional CID in ion traps. CID and IRMPD using short irradiation times of duplex siRNA resulted in strand separation, similar to the dissociation trends observed for duplex DNA. With longer irradiation times, however, the individual single-strands underwent secondary dissociation to yield informative sequence ions not obtained by CID.     

Electron detachment dissociation of dermatan sulfate oligosaccharides

The structural characterization of glycosaminoglycans (GAG) oligosaccharides has been a long-standing challenge in the field of mass spectrometry. In this work, we present the application of electron detachment dissociation (EDD) Fourier transform mass spectrometry to the analysis of dermatan sulfate (DS) oligosaccharides up to 10 residues long. The EDD mass spectra of DS oligosaccharides were compared with their infrared multiphoton dissociation (IRMPD) mass spectra. EDD produces more abundant fragmentation than IRMPD with far less loss of SO3 from labile sulfate modifications. EDD cleaves all glycosidic bonds, yielding both conventional glycosidic bond fragmentation as well as satellite peaks resulting from the additional loss of 1 or 2 hydrogen atoms. EDD also yields more cross-ring fragmentation than IRMPD. For EDD, abundant cross-ring fragmentation in the form of A- and X-ions is observed, with 1,5Xn cleavages occurring for all IdoA residues and many of the GalNAc4S residues, except at the reducing and nonreducing ends. In contrast, IRMPD produces only A-type cross-ring fragmentation for long oligosaccharides (dp6-dp10). As all the structurally informative fragment ions observed by IRMPD appear as a subset of the peaks found in the EDD mass spectrum, EDD shows great potential for the characterization of GAG oligosaccharides using a single tandem mass spectrometry experiment.     

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.     

Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisional-activated dissociation on a triple stage quadrupole instrument

Structural characterization of glycosphingolipids as their lithiated adducts using low-energy collisional-activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) is described. The tandem mass spectra contain abundant fragment ions reflecting the long chain base (LCB), fatty acid, and the sugar constituent of the molecule and permit unequivocal identification of cerebrosides, di-, trihexosyl ceramides and globosides. The major fragmentation pathways arise from loss of the sugar moiety to yield a lithiated ceramide ion, which undergoes further fragmentation to form multiple fragment ions that confirm the structures of the fatty acid and LCB. The mechanisms for the ion formation and the possible configuration of the fragment ions, resulting from CAD of the lithiated molecular ions ([M + Li]+) of monoglycosylceramides are proposed. The mechanisms were supported by CAD and source CAD tandem mass spectra of various cerebrosides and of their analogous molecules prepared by H-D exchange. Constant neutral loss and precursor ion scannings to identify galactosylceramides with sphingosine or sphinganine LCB subclasses, and with specific N-2-hydroxyl fatty acid subclass in mixtures are also demonstrated.     

Consecutive Fragmentation Mechanisms of Protonated Ferulic Acid Probed by Infrared Multiple Photon Dissociation Spectroscopy and Electronic Structure Calculations

Protonated ferulic acid and its principle fragment ion have been characterized using infrared multiple photon dissociation spectroscopy and electronic structure calculations at the B3LYP/6-311?+?G(d,p) level of theory. Due to its extensively conjugated structure, protonated ferulic acid is observed to yield three stable fragment ions in IRMPD experiments. It is proposed that two parallel fragmentation pathways of protonated ferulic acid are being observed. The first pathway involves proton transfer, resulting in the loss of water and subsequently carbon monoxide, producing fragment ions m/z 177 and 149, respectively. Optimization of m/z 177 yields a species containing an acylium group, which is supported by a diagnostic peak in the IRMPD spectrum at 2168?cm^?1. The second pathway involves an alternate proton transfer leading to loss of methanol and rearrangement to a five-membered ring.     

Peptide and protein quantification using iTRAQ with electron transfer dissociation

Electron transfer dissociation (ETD) has become increasingly used in proteomic analyses due to its complementarity to collision-activated dissociation (CAD) and its ability to sequence peptides with post-translation modifications (PTMs). It was previously unknown, however, whether ETD would be compatible with a commonly employed quantification technique, isobaric tags for relative and absolute quantification (iTRAQ), since the fragmentation mechanisms and pathways of ETD differ significantly from CAD. We demonstrate here that ETD of iTRAQ labeled peptides produces c- and z -type fragment ions as well as reporter ions that are unique from those produced by CAD. Exact molecular formulas of product ions were determined by ETD fragmentation of iTRAQ-labeled synthetic peptides followed by high mass accuracy orbitrap mass analysis. These experiments revealed that ETD cleavage of the N-C(alpha) bond of the iTRAQ tag results in fragment ions that could be used for quantification. Synthetic peptide work demonstrates that these fragment ions provide up to three channels of quantification and that the quality is similar to that provided by beam-type CAD. Protein standards were used to evaluate peptide and protein quantification of iTRAQ labeling in conjunction with ETD, beam-type CAD, and pulsed Q dissociation (PQD) on a hybrid ion trap-orbitrap mass spectrometer. For reporter ion intensities above a certain threshold all three strategies provided reliable peptide quantification (average error < 10%). Approximately 36%, 8%, and 16% of scans identified fall below this threshold for ETD, HCD, and PQD, respectively. At the protein level, average errors were 2.3%, 1.7%, and 3.6% for ETD, HCD, and PQD, respectively.     

Surface-induced dissociation of singly and multiply protonated polypropylenamine dendrimers

The ease of fragmentation of various charge states of protonated polypropylenamine (POPAM) dendrimers is investigated by surface-induced dissociation. Investigated are the protonated diaminobutane propylenamines [DAB(PA)n] DAB(PA)8 (1+ and 2+), DAB(PA)16 (2+ and 3+), and DAB(PA)32 (3+ and 4+). These ions have been proposed to fragment by charge-directed intramolecular nucleophilic substitution (SNi) reactions. Differences in relative fragment ion abundances between charge states can be related to the occupation of different protonation sites. These positions can be rationalized based on estimates of Coulomb energies and gas-phase basicities of the protonation/fragmentation sites. The laboratory collision energies at which the fragment ion current is approximately 50% of the total ion current were found to increase with the size, but to be independent of charge state of the protonated POPAM dendrimers. It is suggested that intramolecular Coulomb repulsion within the multiply protonated POPAM dendrimers selected for activation does not readily result in easier fragmentation, which is in accordance with the proposed fragmentation mechanism.