Tandem mass spectrometry investigation of ADP-ribosylated kemptide

Bacterial adenosine diphosphate-ribosyltransferases (ADPRTs) are toxins that play a significant role in pathogenicity by inactivating host proteins through covalent addition of ADP-ribose. In this study we used ADP-ribosylated Kemptide (LRRASLG) as a standard to examine the effectiveness of three common tandem mass spectrometry fragmentation methods for assignment of amino acid sequence and site of modification. Fragmentation mechanisms investigated include low-energy collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron-capture dissociation (ECD); all were performed on a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. We show that ECD, but neither CID nor IRMPD, of ADP-ribosylated Kemptide produces tandem mass spectra that are interpretable with regard to amino acid sequence assignment and site of modification. Examination of CID and IRMPD tandem mass spectra of ADP-ribosylated Kemptide revealed that fragmentation was primarily focused to the ADP-ribose region, generating several potential diagnostic ions for use in discovery of ADP-ribosylated proteins. Because of the lower relative sensitivity of ECD during data-dependent acquisition to CID, we suggest a 2-fold strategy where CID and IRMPD are first used to detect ADP-ribosylated peptides, followed by sequence assignment and location of modification by ECD analysis.     

Implementation of dipolar direct current (DDC) collision-induced dissociation in storage and transmission modes on a quadrupole/time-of-flight tandem mass spectrometer

Means for effecting dipolar direct current collision-induced dissociation (DDC CID) on a quadrupole/time-of-flight in a mass spectrometer have been implemented for the broadband dissociation of a wide range of analyte ions. The DDC fragmentation method in electrodynamic storage and transmission devices provides a means for inducing fragmentation of ions over a large mass-to-charge range simultaneously. It can be effected within an ion storage step in a quadrupole collision cell that is operated as a linear ion trap or as ions are continuously transmitted through the collision cell. A DDC potential is applied across one pair of rods in the quadrupole collision cell of a QqTOF hybrid mass spectrometer to effect fragmentation. In this study, ions derived from a small drug molecule, a model peptide, a small protein, and an oligonucleotide were subjected to the DDC CID method in either an ion trapping or an ion transmission mode (or both). Several key experimental parameters that affect DDC CID results, such as time, voltage, low mass cutoff, and bath gas pressure, are illustrated with protonated leucine enkephalin. The DDC CID dissociation method gives a readily tunable, broadband tool for probing the primary structures of a wide range of analyte ions. The method provides an alternative to the narrow resonance conditions of conventional ion trap CID and it can access more extensive sequential fragmentation, depending upon conditions. The DDC CID approach constitutes a collision analog to infrared multiphoton dissociation (IRMPD).     

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.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.     

New aspects in fragmentation of peptide nucleic acids: comparison of positive and negative ions by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The use of peptide nucleic acids (PNAs) is steadily increasing in biochemistry and diagnostics. So far, PNAs have mostly been investigated using cationic conditions in mass spectrometry. Furthermore, the use of fragmentation techniques developed for peptides and proteins like infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) has barely been examined. However, especially the fragmentation behavior of PNA oligomers in negative ion mode is of high importance, due to the ability to interact with nucleic acids which are almost exclusively analyzed in the negatively charged state. In the current study PNA fragmentations under cationic and anionic conditions were investigated and different fragmentation techniques like collision‐induced dissociation (CID), IRMPD and ECD were applied. Especially when using CID and IRMPD, amide bonds were broken, whereas ECD resulted in the elimination of nucleobases. Differences were also observed between positive and negative ionization, while the sequence coverage for the negative ions was superior to positive ions. The fragmentation behavior using IRMPD led to almost complete sequence coverage. Additionally, in anions the interesting effect of multiple eliminations of HNCO was found. Copyright © 2009 John Wiley & Sons, Ltd.     

Enhancement of ion transmission at low collision energies via modifications to the interface region of a spectrometer

The transmission efficiency of precursor and product ions decreases significantly at lower collision energies in a four-sector tandem mass spectrometer. In an effort to improve the overall ion transmission in this energy regime three modifications were made in the interface region between the two stages of mass analysis. An einzel lens was inserted prior to the deceleration lens of the collision cell block to reduce the precursor ion beam diameter. The collision cel1 block was reduced in thickness while maintaining the collision path length, thus increasing the number of ions which entered and exited the gas chamber, while removing any stray electrical fields. Finally, a second active focusing element was incorporated after the collision cell block to enhance the collection efficiency of the product ions. A tandem mass spectrum of angiotensin I obtained with this interface, at a collision cell block potential of 9200 volts, exhibited classical high energy collision-induced dissociation (CID) fragmentation patterns, a precursor ion transmission of 92% and an overall CID efficiency of approximately 7.5%. These improvements have resulted in a dramatically higher overall ion transmission at high collision cell potentials as well as sufficient sensitivity in acquiring good quality CID spectra in the lower collision energy regime (i.e., 60 eV). (460-469)     

Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

The analytical utility of the electron capture dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.     

Comparison of the activation time effects and the internal energy distributions for the CID,PQD and HCD excitation modes

Reproducibility among different types of excitation modes is a major bottleneck in the field of tandem mass spectrometry library development in metabolomics. In this study, we specifically evaluated the influence of collision voltage and activation time parameters on tandem mass spectrometry spectra for various excitation modes [collision‐induced dissociation (CID), pulsed Q dissociation (PQD) and higher‐energy collision dissociation (HCD)] of Orbitrap‐based instruments. For this purpose, internal energy deposition was probed using an approach based on Rice–Rampserger–Kassel–Marcus modeling with three thermometer compounds of different degree of freedom (69, 228 and 420) and a thermal model. This model treats consecutively the activation and decomposition steps, and the survival precursor ion populations are characterized by truncated Maxwell–Boltzmann internal energy distributions. This study demonstrates that the activation time has a significant impact on MS/MS spectra using the CID and PQD modes. The proposed model seems suitable to describe the multiple collision regime in the PQD and HCD modes. Linear relationships between mean internal energy and collision voltage are shown for the latter modes and the three thermometer molecules. These results suggest that a calibration based on the collision voltage should provide reproducible for PQD, HCD to be compared with CID in tandem in space instruments. However, an important signal loss is observed in PQD excitation mode whatever the mass of the studied compounds, which may affect not only parent ions but also fragment ions depending on the fragmentation parameters. A calibration approach for the CID mode based on the variation of activation time parameter is more appropriate than one based on collision voltage. In fact, the activation time parameter in CID induces a modification of the collisional regime and thus helps control the orientation of the fragmentation pathways (competitive or consecutive dissociations). Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of in‐source collision‐induced dissociation and beam‐type collision‐induced dissociation of emerging synthetic drugs using a high‐resolution quadrupole time‐of‐flight mass spectrometer

In‐source collision‐induced dissociation (CID) is commonly used with single‐stage high‐resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in‐source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in‐source CID and beam‐type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in‐source CID. Electrospray ionization–quadrupole time‐of‐flight (ESI‐Q‐TOF) mass spectrometry was used to generate product ion spectra from in‐source CID and beam‐type CID for a series of well‐characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in‐source CID and a range of collision energies for beam‐type CID. The results indicate that large fragmentor potentials for in‐source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam‐type CID. Although conditions can be found in which in‐source CID and beam‐type CID provide similar overall spectra, the in‐source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam‐type CID.     

Electron capture dissociation proceeds with a low degree of intramolecular migration of peptide amide hydrogens

Hydrogen (1H/2H) exchange combined with mass spectrometry (HX-MS) has become a recognized method for the analysis of protein structural dynamics. Presently, the incorporated deuterons are typically localized by enzymatic cleavage of the labeled proteins and single residue resolution is normally only obtained for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation in tandem mass spectrometers would greatly increase the applicability of the HX-MS method. The biggest obstacle in achieving this goal is the intramolecular hydrogen migration (i.e., hydrogen scrambling) that occurs during vibrational excitation of gas-phase ions. Unlike traditional collisional ion activation, electron capture dissociation (ECD) is not associated with substantial vibrational excitation. We investigated the extent of intramolecular backbone amide hydrogen (1H/2H) migration upon ECD using peptides with a unique selective deuterium incorporation. Our results show that only limited amide hydrogen migration occurs upon ECD, provided that vibrational excitation prior to the electron capture event is minimized. Peptide ions that are excessively vibrationally excited in the electrospray ion source by, e.g., high declustering potentials or during precursor ion selection (via sideband excitation) in the external linear quadrupole ion trap undergo nearly complete hydrogen (1H/2H) scrambling. Similarly, collision-induced dissociation (CID) in the external linear quadrupole ion trap results in complete or extensive hydrogen (1H/2H) scrambling. This precludes the use of CID as a method to obtain site-specific information from proteins that are labeled in solution-phase 1H/2H exchange experiments. In contrast, the deuteration levels of the c- and z-fragment ions generated from ECD closely mimic the known solution deuteration pattern of the selectively labeled peptides. This excellent correlation between the results obtained from gas phase and solution suggests that ECD holds great promise as a general method to obtain single residue resolution in proteins from solution 1H/2H exchange experiments.     

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.     

Energy‐dependent normal and unusually large inverse chlorine kinetic isotope effects of simple chlorohydrocarbons in collision‐induced dissociation by gas chromatography‐electron ionization‐tandem mass spectrometry

Kinetic isotope effects (KIEs) occurring in mass spectrometry (MS) can provide in‐depth insights into the fragmentation behaviors of compounds of interest in MS. Yet, the fundamentals of KIEs in collision‐induced dissociation (CID) in tandem mass spectrometry (MS/MS) are unclear, and information about chlorine KIEs (Cl‐KIEs) of organochlorines in MS is particularly scarce. This study investigated the Cl‐KIEs of dichloromethane, trichloroethylene, and tetrachloroethylene during CID using gas chromatography‐electron ionization triple‐quadrupole MS/MS. Cl‐KIEs were evaluated with MS signal intensities. All the organochlorines presented large inverse Cl‐KIEs (<1, the departures of Cl‐KIEs from 1 denote the magnitudes of Cl‐KIEs), showing the largest magnitudes of 0.797, 0.910, and 0.892 at the highest collision energy (60 eV) for dichloromethane, trichloroethylene, and tetrachloroethylene, respectively. For dichloromethane, both intra‐ion and inter‐ion Cl‐KIEs were studied, within the ranges of 0.820–1.020 and 0.797–1.016, respectively, showing both normal and inverse Cl‐KIEs depending on collision energies. The observed Cl‐KIEs generally declined from large normal to extremely large inverse values with increasing collision energies from 0 to 60 eV but were inferred to be independent of MS signal intensities. The Cl‐KIEs are dominated by critical energies at low internal energies of precursor ions, resulting in normal Cl‐KIEs; while at high internal energies, the Cl‐KIEs are controlled by rotational barriers (or looseness/tightness of transition states), which lead to isotope‐competitive reactions in dechlorination and thereby inverse Cl‐KIEs. It is concluded that the Cl‐KIEs may depend on critical energies, bond strengths, available internal energies, and transition state looseness/tightness. The findings of this study yield new insights into the fundamentals of Cl‐KIEs of organochlorines during CID and may be conducive to elucidating the underlying mechanisms of KIEs in collision‐induced and photo‐induced reactions in the actual world.     

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.     

Fragmentation of Singly,Doubly, and Triply Charged Hydrogen Deficient Peptide Radical Cations in Infrared Multiphoton Dissociation and Electron Induced Dissociation

Gas phase fragmentation of hydrogen deficient peptide radical cations continues to be an active area of research. While collision induced dissociation (CID) of singly charged species is widely examined, dissociation channels of singly and multiply charged radical cations in infrared multiphoton dissociation (IRMPD) and electron induced dissociation (EID) have not been, so far, investigated. Here, we report on the gas phase dissociation of singly, doubly and triply charged hydrogen deficient peptide radicals, [M + nH]^(n+1)+· (n = 0, 1, 2), in MS³ IRMPD and EID and compare the observed fragmentation pathways to those obtained in MS³ CID. Backbone fragmentation in MS³ IRMPD and EID was highly dependent on the charge state of the radical precursor ions, whereas amino acid side chain cleavages were largely independent of the charge state selected for fragmentation. Cleavages at aromatic amino acids, either through side chain loss or backbone fragmentation, were significantly enhanced over other dissociation channels. For singly charged species, the MS³ IRMPD and EID spectra were mainly governed by radical-driven dissociation. Fragmentation of doubly and triply charged radical cations proceeded through both radical- and charge-driven processes, resulting in the formation of a wide range of backbone product ions including, a-, b-, c-, y-, x-, and z-type. While similarities existed between MS³ CID, IRMPD, and EID of the same species, several backbone product ions and side chain losses were unique for each activation method. Furthermore, dominant dissociation pathways in each spectrum were dependent on ion activation method, amino acid composition, and charge state selected for fragmentation.     

Dissociation of biomolecules using a ultraviolet matrix-assisted laser desorption/ionisation time-of-flight/curved field reflectron tandem mass spectrometer equipped with a differential-pumped collision cell

A commercial matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) instrument equipped with a curved field reflectron (CFR) was modified in order to perform collision-induced dissociation (CID) on a variety of biomolecules. The incorporation of a high-resolution ion gate together with a collision cell within the field-free region allowed tandem mass analysis (MS/MS), without the necessity to decelerate the precursor ions prior to activation. The simultaneous detection of all product ions remained possible by using the CFR. To test the MS/MS performances, ACTH (fragment 1-17), a complex high mannose carbohydrate (Man)(8)(GlcNac)(2) and a lysophosphatidylcholine lipid (18:1) were analysed on the modified instrument. Direct comparison with the low-energy product ion spectra, acquired on a MALDI quadrupole ion trap (QIT) two-stage reflectron time-of flight (ReToF) mass spectrometer, showed significant differences in the types of product ions observed. The additional ions detected were a clear indication of the high-energy fragmentation processes occurring in the collision cell.     

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)-rich and phosphorothioate (PS)-modified DNA oligonucleotide was investigated using two fragmentation techniques: trap-type collision-induced dissociation (“CID”) and beam-type CID also termed as higher-energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC-MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T-rich DNA oligonucleotides. Specifically, HCD provided more sequence-defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS-modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T-rich PS-modified DNA oligonucleotides.     

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.     

Axial CID and high pressure resonance CID in a miniature ion trap mass spectrometer using a discontinuous atmospheric pressure interface

Axial collision induced dissociation (CID) and high-pressure resonance CID were implemented and compared with normal low-pressure resonance CID in a miniature ion trap mass spectrometer to obtain more complete fragmentation spectra. Axial CID was realized simply by applying a potential to the discontinuous atmospheric pressure interface (DAPI) capillary without performing parent ion isolation before dissociation. High-pressure resonance CID employed a double-introduction pulse scan function, by means of which precursor ions isolated at low-pressure (<10⁻³ torr) were dissociated at high-pressure (0.1 torr-1 torr) with higher excitation energy, so that tandem MS of isolated precursor ions was achieved and extensive fragmentation was obtained. A simple peptide (Leu-enkephalin) and dye molecule (rhodamine B) ionized by ESI were used to investigate both methods and compare them with normal low-pressure resonance CID.     

Electron capture dissociation mass spectrometry of metallo-supramolecular complexes

The electron capture dissociation (ECD) of metallo-supramolecular dinuclear triple-stranded helicate Fe₂L₃⁴⁺ ions was determined by Fourier transform ion cyclotron resonance mass spectrometry. Initial electron capture by the di-iron(II) triple helicate ions produces dinuclear double-stranded complexes analogous to those seen in solution with the monocationic metal centers Cu^I or Ag^I. The gas-phase fragmentation behavior [ECD, collision-induced dissociation (CID), and infrared multiphoton dissociation (IRMPD)] of the di-iron double-stranded complexes, (i.e., MS³ of the ECD product) was compared with the ECD, CID, and IRMPD of the Cu^I and Ag^I complexes generated from solution. The results suggest that iron-bound dimers may be of the form Fe₂^IL₂²⁺ and that ECD by metallo-complexes allows access, in the gas phase, to oxidation states and coordination chemistry that cannot be accessed in solution.     

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.