Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]¹⁵⁺ ion being the most intense peak. With the addition of H₂O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.     

Observation and implications of high mass-to-charge ratio ions from electrospray ionization mass spectrometry

High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (M _r 12,231) and porcine pepsin (M _r 34,584), by using a quadrupole mass spectrometer with an m/z 45,000 range. The ESI mass spectrum for cytochrome c in an aqueous solution gives a charge state distribution that ranges from 12 + to 2 +, with a broad, low-intensity peak in the mass-to-charge ratio region corresponding to the [M + H]⁺ ion. the negative ion ESI mass spectrum for pepsin in 1% acetic acid solution shows a charge state distribution ranging from 7? to 2?. To observe the [M - H]^? ion, harsher desolvation and interface conditions were required. Also observed was the abundant aggregation of the protens with average charge states substantially lower than observed for their monomeric counterparts. The negative ion ESI mass spectrum for cytochrome c in 1–100 mM NH₄OAc solutions showed greater relative abundances for the higher mass-to-charge ratio ions than in acuidic solutions, with an [M - H]^? ion relative abundance approximately 50% that of the most abundant charge state peak. The observation that protein aggregates are formed with charge states comparable to monomeric species (at fower mass-to-charge ratios) suggests that the high mass-to-charge ratio monomers may be formed by the dissociation of aggregate species. The observation of low charge state and aggregate molecular ions concurrently with highly charged species may serve to support a variation of the charged residue model, originally described by Dole and co-workers (Dole, M., et al. J. Chem. Phys. 1968, 49, 2240; Mack, L. L., et al. J. Chem. Phys. 1970, 52, 4977) which involves the Coulombically driven formation of either very highly solvated molecular ions or lower ananometer-diameter droplets.     

Coupling electrospray corona discharge, charge reduction and ion mobility mass spectrometry: From peptides to large macromolecular protein complexes

We present the design and implementation of a home-built point-to-plane corona discharge probe, which rapidly and efficiently charge reduces biological ions generated by electrospray ionization (ESI). The molecules analysed ranged from small peptides such as Glu-fibrinopeptide B (1.5 kDa), small proteins such as myoglobin (16.9 kDa), polymers such as polyethylene glycol (PEG 10 k) which all showed intense singly charged ions; to large native multiprotein complexes such as GroEL (802 kDa) which show a broad range of charge-reduced species. The corona discharge probe operates at atmospheric pressure and was directly interfaced with a standard-ESI or nanoflow-ESI source of quadrupole ion mobility time-of-flight mass spectrometer. The corona discharge probe is completely modular and could potentially be mounted to any commercial or research grade mass spectrometer with an ESI source. The level of charge reduction is precisely controlled by the applied voltage and/or probe gas flow rate and when in operation, results in approximately a 50 % reduction in total ion current. We also present the combination of corona discharge and travelling wave ion mobility and assign helium collision cross-section values (Ω_He) to the charge reduced species of the native protein complex pyruvate kinase. It would appear that the Ω_He of the +20 charge state for pyruvate kinase is approximately 20 % smaller than the +35 charge state. Finally, we discuss the potential benefits and concerns of utilising charge reduced protein species as a means of extending the travelling wave collision cross-section calibration range over that which is already published.     

"Dueling" ESI: instrumentation to study ion/ion reactions of electrospray-generated cations and anions

Novel instrumentation has been developed which allows for the sequential injection and subsequent reaction of oppositely-charged ions generated via electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer. The instrument uses a DC turning quadrupole to sequentially direct the two ion polarities into the ion trap from ESI sources which are situated 90 degrees from the axial (z) dimension of the trap, and 180 degrees from one another. This arrangement significantly expands the range of ionic reactants amenable to study over previously-used instrumentation. For example, ion/ion reactions of multiply-charged positive ions with multiply-charged negative ions can be studied. Also, reactions of multiply-charged ions with singly-charged ions of opposite polarity that could not be generated by previously used ionization methods, or that could not be efficiently injected through the ion trap ring electrode, can be studied with the new instrument. This capability allows, for example, the charge state manipulation of negatively-charged precursor and product ions derived from proteins and oligonucleotides via proton transfer reactions with singly-charged cations generated by ESI.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Mechanistic studies of multipole storage assisted dissociation

The degree and onset of fragmentation in multipole storage assisted dissociation (MSAD) have been investigated as functions of several hexapole parameters. Strict studies of hexapole charge density (number of ions injected) and hexapole storage time were made possible by placing a pulsed shutter in front of the entrance to the mass spectrometer. The results obtained show that the charge density is the most critical parameter, but also dependencies on storage time, radio-frequency (rf) -amplitude, and pressure are seen. From these data, and from simulations of the ion trajectories inside the hexapole, a mechanism for MSAD, similar to the ones for sustained off-resonance irradiation (SORI), and for low energy collisionally induced dissociation in the collision multipole of a triple quadrupole mass spectrometer, is proposed. It is believed that, at higher charge densities, ions are pushed to larger hexapole radii where the electric potential created by the rf field is higher, forcing the ions to oscillate radially to higher amplitudes and thereby reach higher (but still relatively low) kinetic energies. Multiple collisions with residual gas molecules at these elevated energies then heat up the molecules to their dissociation threshold. Further support for this mechanism is obtained from a comparison of MSAD and SORI spectra which are almost identical in appearance.     

Ligand displacement reactions of dimer and trimer pyridyl ligand/transition metal complexes

Ligand exchange reactions of pyridyl ligand/transition metal complexes are examined in a quadrupole ion trap mass spectrometer to evaluate the ability of multidentate ligands to displace other pyridyl ligands in complexes where the charge is highly delocalized and there is a great degree of ligand repulsions. Partially or fully coordinated transition metal ions in dimer or trimer species involving small mono- or bidentate pyridyl ligands undergo ligand displacement reactions with larger bi- and tridentate pyridyl ligands. Larger ligands with greater chelation abilities, such as 1,10-phenanthroline and 2,2′:6,2″-terpyridine, are often able to simultaneously displace two nonchelating ligands from a partially coordinated metal ion. However, the analogous reactions involving displacement of bidentate chelating ligands from more fully coordinated transition metal ion complexes are nearly quenched. In other cases, mixed-ligand dimer and trimer complexes are observed, indicating step-wise displacement of the initially complexed ligands.     

Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B(5) homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60 degrees C and subunit concentrations ranging from 5 to 85 microM, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 microM, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B(5) ions of Stx2 at the +12 and +13 charge states proceeds, at reaction temperatures of 120 to 180 degrees C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B(5) ions at the +12 and +13 charge states were compared with values reported for the corresponding B(5) ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (E(a)) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the E(a) values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins.     

Practical considerations when using radio frequency-only quadrupole ion guide for atmospheric pressure ionization sources with time-of-flight mass spectrometry

Construction details and performance evaluation of a radio frequency (rf)-only quadrupole ion guide for use with an electrospray ionization time-of-flight mass spectrometer is presented in this paper. Angiotensin III and cytochrome c were used in these experiments to investigate the ion transmission properties of the rf-only quadrupole for different m/z species. In addition, influence of ion kinetic energies along with the characteristic fragmentation due to collision induced dissociation (CID) were studied. These experiments demonstrate that the transmissions of different m/z ions were not only dependent on the frequency and magnitude of the rf waveform, which is similar to a high vacuum rf-only quadrupole ion guide, but also on the pressure inside the quadrupole chamber. For the pressure range tested, low m/z ions are better focused with increasing pressure. As expected, transmission of ions are subject to space charge limitations when significant numbers of ions are focused on the axis of the quadrupole. It is also observed that CID results are related to transverse motion and longitude motion of ions inside the quadrupole region. Consequently, CID is useful for fragmentation of linear peptides and it is not effective (in present configuration) for large bulky proteins. The kinetic energy of ions that enter the repelling region of the TOFMS is ultimately determined by the ensemble effect resulting from the dc bias potential of the quadrupole (the dominant factor), skimmer-2, pressure inside the quadrupole chamber, and jet expansion. While this system is tested with an ESI source, the operational principle and design criteria are directly applicable for improving other atmospheric pressure ionization sources with time-of-flight mass analyzers such as an inductively coupled plasma ion source.     

Fluorescence resonance energy transfer in gaseous, mass-selected polyproline peptides

Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.     

Protein structural effects in gas phase ion/molecule reactions with diethylamine.

The relationship between gas-phase protein structure and ion/molecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressure inlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of 'native' proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.     

Structural studies on Helicobacter pylori 3‐deoxy‐D‐manno‐2‐octulosonate‐8‐phosphate synthase using electrospray ionization mass spectrometry: a tetrameric complex composed of dimeric dimers

Helicobacter pylori 3‐deoxy‐D ‐manno‐2‐octulosonate‐8‐phosphate (KDO8P) synthase catalyzes the conversion of D ‐arabinose‐5‐phosphate (A5P) and phosphoenolpyruvate (PEP) to produce KDO8P and inorganic phosphate. Since this protein is absent in mammals, it might therefore be an attractive target for the development of new antibiotics. Unlike E. coli KDO8P synthase (class I), the H. pylori counterpart is a class II enzyme, where it requires a divalent transition metal ion for catalysis. Although the metal ions have been shown to be important for catalysis, their role in the structure is not understood. Using electrospray ionization mass spectrometry (ESI‐MS), the role of the metal ions in H. pylori KDO8P synthase has been investigated. This protein is found to be a tetramer in the gas phase but dissociates into the dimer with increasing declustering potential (DP2) suggesting an existence of a ‘structurally specific’ tetramer. An examination of mass spectra revealed that the tetrameric state of the Cd²⁺‐reconstituted enzyme is less stable than those of the Zn²⁺‐, Co²⁺‐ and Cu²⁺‐enzymes. The stoichiometry of metal binding to the protein depends on the nature of the metal ion. Taken together, our data suggest that divalent metal ions play an important role in the quaternary structure of the protein and the tetrameric state may be primarily responsible for catalysis. This study demonstrates the first structural characterization and stoichiometry of metal binding in class II KDO8P synthase using electrospray ionization quadrupole time‐of‐flight mass spectrometry under nondenaturing conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Sources of artefacts in the electrospray ionization mass spectra of saturated diacylglycerophosphocholines: From condensed phase hydrolysis reactions through to gas phase intercluster reactions

The mass spectra of diacylglycerophosphocholine phospholipids comprised of saturated fatty acids (1,2-dipentanoyl-sn-glycero-3-phosphocholine (D5PC); 1,2-dihexanoyl-sn-glycero-3-phosphocholine (D6PC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (D14PC)) are sensitive to the electrospray ionization (ESI) conditions. When fresh solutions of phospholipid in 10 mM ammonium acetate are subjected to ESI, protonated oligomeric clusters, [DxPCn + H]+ (x = 5, 6, and 14) are observed in the following different types of mass spectrometers: 3D-quadrupole ion trap; linear ion trap, and triple quadrupole. The formation of the protonated cluster ions is not unique to the ion trap instruments, although they tend to be more abundant in these instruments. As the ESI solutions age, new ions are observed, which correspond to acid-catalyzed solution phase deacylation reactions. The collision induced dissociation fragmentation reactions of the oligomer cluster ions exhibit a distinct dependence on the cluster size, with the larger clusters (n > 2) simply fragmenting via the loss of lipid monomers. In contrast, the fragmentation of the dimeric cluster ion is unique, resulting in a number of additional reactions including covalent bond formation via intermolecular cluster SN2 reactions and SN2 transfer of a methyl group. The nature of the charge has a significant role in the formation of products via these intermolecular cluster reactions. Changing the head group to phosphoethanolamine "switches off" the SN2 reactions, while changing the cation from a proton to either a sodium or a potassium ion, diminishes the intermolecular reactions relative to monomer loss. Semi empirical PM3 calculations on [D6PC2 + H]+ suggest that the SN2 reactions are thermodynamically favored over simple monomer loss. These results have important implications in the field of lipidomics.     

Atmospheric pressure ionization mass spectrometry techniques for the analysis of alkyl ethoxysulfate mixtures

This paper compares two liquid introduction atmospheric pressure ionization techniques for the analysis of alkyl ethoxysulfate (AES) anionic surfactant mixtures by mass spectrometry, i. e., electrospray ionization (ESI) in both positive and negative ion modes and atmospheric pressure chemical ionization (APCI) in positive ion mode, using a triple quadrupole mass spectrometer. Two ions are observed in ESI(+) for each individual AES component, [M + Na]⁺ and a “desulfated” ion [M − SO₃ + H]⁺, whereas only one ion, [M − Na]⁻ is observed for each AES component in ESI(−). APCI(+) produces a protonated, “desulfated” ion of the form [M − NaSO₃ + 2H]⁺ for each AES species in the mixture under low cone voltage (10 V) conditions. The mass spectral ion intensities of the individual AES components in either the series from ESI(+) or APCI(+) can be used to obtain an estimate of their relative concentrations in the mixture and of the average ethoxylate (EO) number of the sample. The precursor ions produced by either ESI(+) or ESI(−), when subjected to low-energy (50 eV) collision-induced dissociation, do not fragment to give ions that provide much structural information. The protonated, desulfated ions produced by APCI(+) form fragment ions which reveal structural information about the precursor ions, including alkyl chain length and EO number, under similar conditions. APCI(+) is less susceptible to matrix effects for quantitative work than ESI(+). Thus APCI(+) provides an additional tool for the analysis of anionic surfactants such as AES, especially in complex mixtures where tandem mass spectrometry is required for the identification of the individual components.     

Observation of noncovalent complexes to the avidin tetramer by electrospray ionization mass spectrometry

Intact avidin-biotin and avidin-biotin maleimide noncovalent complexes have been observed by electrospray ionization mass spectrometry (ESI-MS) by using an extended mass range quadrupole mass spectrometer. By utilizing mild ES1 interface conditions, the expected solution behavior of four biotin or biotin maleimide molecules noncovalently binding to each avidin tetramer can be preserved in the gas phase. The ESI-MS results show the appropriate mass additions of 973 ± 60 Da for biotin and 1802 ± 40 Da for biotin maleimide to the avidin tetramer species. These results support the hypothesis that substantial retention of higher order structure is possible in the gas phase by using gentle ESI conditions.     

Transmission mode ion/ion reactions in the radiofrequency‐only ion guide of hybrid tandem mass spectrometers

Transmission mode ion/ion reactions have been performed within the first quadrupole, the Q0 radiofrequency (RF)‐only quadrupole, of two types of hybrid tandem mass spectrometers (viz., triple quadrupole/linear ion trap and QqTOF instruments). These transmission mode reactions involved the storage of either the reagent species and the transmission of the analyte species through the Q0 quadrupole for charge inversion reactions or the storage of the analyte ions and transmission of the reagent ions as in charge reduction experiments. A key advantage to the use of transmission mode ion/ion reactions is that they do not require any instrument hardware modifications to provide interactions of oppositely charged ions and can be implemented in any instrument that contains a quadrupole or linear ion trap. The focus of this work was to investigate the potential of using the RF‐only quadrupole ion guide positioned prior to the first mass‐resolving element in a tandem mass spectrometer for ion/ion reactions. Two types of exemplary experiments have been demonstrated. One involved a charge inversion reaction and the other involved a charge reduction reaction in conjunction with ion parking. Ion/ion reactions proved to be readily implemented in Q0 thereby adding significantly greater experimental flexibility in the use of ion/ion reaction experiments with hybrid tandem mass spectrometers. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospray Ionization Tandem Mass Spectrometry of Ammonium Cationized Polyethers

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine’s degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Study of diketone/metal ion complexes by electrospray ionization mass spectrometry: Influence of Keto-Enol tautomerism and chelation

The formation and collisionally activated dissociation (CAD) behavior of a series of complexes containing cyclic or linear diketone ligands and alkali, alkaline earth, or transition metal ions are investigated. Electrospray ionization (ESI) is utilized for introduction of the metal ion complexes into a quadrupole ion trap mass spectrometer. The proximity of the carbonyl groups is crucial for formation and detection of ion complexes by ESI. For example, no metal ion complexes are observed for 1,4-cyclohexanedione, but they are readily detected for the isomers, 1,2-and 1,3-cyclohexanedione. Although the diketones form stable doubly charged complexes, the formation of singly charged alkaline earth complexes of the type (nL + M²⁺ ? H⁺)⁺ where L = 1,3-cyclohexanedione or 2,4-pentanedione is the first evidence of charge reduction. CAD investigations provide further evidence of charge reduction processes occurring in the gas-phase complexes. The CAD studies indicate that an intramolecular proton transfer between two diketone ligands attached to a doubly charged metal ion, followed by elimination of the resulting protonated ligand, produces the charge reduced complex. For transition metal complexation, the preference for formation of doubly charged versus singly charged complexes correlates with the keto-enol distribution of the diketones in solution.