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

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

Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach.     

Membrane-bound conformation of peptaibols with methyl-deuterated alpha-amino isobutyric acids by 2H magic angle spinning solid-state NMR spectroscopy

We present the use of 2H magic-angle spinning (MAS) NMR on methyl-deuterated alpha-amino isobutyric acid (Aib) as a new method to obtain fast and accurate structural constraints on peptaibols in membrane-bound environments. Using nonoriented vesicle-reconstituted samples we avoid the delicate preparation of oriented samples, and the use of MAS ensures high sensitivity and thereby very fast acquisition of experimental spectra. Furthermore, given the high content ( approximately 40%) of Aib in peptaibols and the fact that the amino acid Aib may be synthesized from cheap starting materials, even in the case of 2H, 13C, or 15N labeling, this method is ideally suited for studies of the membrane-bound conformation of peptaibols.     

Mass spectra of doubly charged ions

The mass spectra of biological molecules, whose molecular mass exceeds 10 kDa, invariably contain multiply charged ions. For example, a survey scan of a small protein will produce singly, doubly and triply protonated molecules, the intensity of the doubly charged species often being greater than that of the singly charged entity. Although the spectra resulting from doubly charged peptides have not previously been studied, collisional activation of such doubly charged species may result in significant additional information pertaining to molecular structure. The techniques employed to study ions originating from multiply charged species were linked scanning of constant B/E and tandem mass spectrometry, namely low collision energy spectra acquired on a BEQQ hybrid instrument. The methodology was applied to model compounds whose tandem mass spectrometry characteristics are well known, e.g. gramicidin S and angiotensin I. The results for the product ions of the [M + 2H]²⁺ species of the models were obtained which highlight the methodology required for high-mass materials.     

Ion mobility augments the utility of mass spectrometry in the identification of human hemoglobin variants

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.     

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.     

Neutral loss of amino acid residues from protonated peptides in collision-induced dissociation generates N- or C-terminal sequence ladders

The widespread occurrence of the neutral loss of one to six amino acid residues as neutral fragments from doubly protonated tryptic peptides is documented for 23 peptides with individual sequences. Neutral loss of amino acids from the N-terminus of doubly charged tryptic peptides results in doubly charged y-ions, forming a ladder-like series with the ions [M + 2H](2+) = y(max) (2+), y(max - 1) (2+), y(max - 2) (2+), etc. An internal residue such as histidine, proline, lysine or arginine appears to favor this type of fragmentation, although it was sometimes also observed for peptides without this structure. For doubly protonated non-tryptic peptides with one of these residues at or near the N-terminus, we observed neutral loss from the C-terminus, resulting in a doubly charged b-type ion ladder. The analyses were performed by Q-TOF tandem mass spectrometry, facilitating the recognition of neutral loss ladders by their 2+ charge state and the conversion of the observed mass differences into reliable sequence information. It is shown that the neutral loss of amino acid residues requires low collision offset values, a simple mechanistic explanation based on established fragmentation rules is proposed and the utility of this neutral loss fragmentation pathway as an additional source for dependable peptide sequence information is documented.     

New short peptaibols from a marine Trichoderma strain

The production of peptaibols by a marine-related Trichoderma longibrachiatum strain was studied using electrospray ionisation multiple-stage ion trap mass spectrometry (ESI-MSn-IT) and gas chromatography/electron impact mass spectrometry (GC/EI-MS). Two major groups of peptaibols were identified, those with long sequences (20 amino acids) and others with short sequences (11 amino acids). This paper describes the methodology used to establish the sequences of short peptaibols in a mixture without previous individual separation. Nine peptaibols were identified. Among them, eight are new, namely as trichobrachin A I-IV (Aib9-Pro10 sequence) and as trichobrachin B I-IV (Val9-Pro10 sequence). Original Pro6-Val7 and Val9-Pro10 sequences have to be noted.     

Decomposition channels for multiply charged ammonia clusters

Multiply charged ammonia cluster ions are produced by adiabatic nozzle expansion and subsequent ionization by electron impact. They are analyzed in a double focussing sector field mass spectrometer (reversed geometry). Doubly charged clusters are only detected above a critical size of 51 and triply charged clusters above 121. Some of these multiply charged ions decay via metastable dissociation processes in the experimental time window accessible. Doubly charged ammonia clusters with sizes ofn≧51 lose one neutral monomer or, roughly ten times less probable, two neutral monomers. Conversely, triply charged ammonia clusters with sizes 110≦n≦120 show an extremely asymmetric Coulomb dissociation resulting in doubly charged cluster ions of about 90% of the initial mass     

Scanning a triple quadrupole mass spectrometer for doubly charged ions

Charge exchange reactions within a triple quadrupole mass spectrometer characterize doubly charged ions formed in the ion source. Two methods have been developed for identifying the singly charged ions formed from doubly charged ions by charge exchange in the collision quadrupole. The first is based on the characteristically high kinetic energy-to-charge ratios of the products of charge exchange; this property can be used to separate these ions from all other singly charged ions. This retarding potential method is analogous to procedures for recording doubly charged ion mass spectra using sector instruments. The second method is based on the fact that, although mass remains constant in the charge exchange reaction, the change in mass-to-charge ratio can be followed. A charge exchange linked scan, predicated on changes in charge rather than mass, but otherwise analogous to neutral loss/gain scans, is described. Information on the structure of doubly charged ions can be obtained by recording the fragmentation products of dissociative charge exchange. The utility of the charge exchange linked scan for the selective identification of polynuclear aromatic compounds in a complex mixture is described. The methods given can be generalized to cover other charge permutation reactions.     

Capillary electrophoresis-electrospray mass spectra of the herbicides paraquat and diquat

The positive ion electrospray mass spectra of the quaternary ammonium salt herbicides paraquat and diquat are examined by on-line separation with capillary electrophoresis (CE) and by direct infusion of the analytes. The analytes are separated by CE in 7–10 min at pH 3.9 in 50% methanol-water by using several different separation buffer electrolytes. The capillary electrophoresis-electrospray ionization (CE-ES) mass spectra of paraquat and diquat consist primarily of doubly charged molecular ions, singly charged molecular ions, and singly charged deprotonated ions. The direct infusion spectra consist primarily of doubly charged molecular ions and singly charged deprotonated ions. The relative abundances of the doubly charged and deprotonated ions depend strongly on the presence or absence of ammonium ion in the CE separation buffer or the direct infusion solution. A deprotonation mechanism is proposed in which the free base ammonia is the deprotonating agent in the desolvating charged droplets or in the gas phase. The analytical potential of the CE-ES electrospray approach for environmental analyses is evaluated in terms of the precision of replicate injections, linear concentration range, and estimated detection limit.     

Effects of the position of internal histidine residues on the collision-induced fragmentation of triply protonated tryptic peptides

The collision-induced dissociation spectra of a series of synthetic, tryptic peptides that differed by the position of an internal histidine residue were studied. Electrospray ionization of these peptides produced both doubly and triply protonated molecular ions. Collision-induced fragmentation of the triply protonated peptide ions had better efficiency than that of the doubly protonated ions, producing a higher abundance of product ions at lower collision energies. The product ion spectra of these triply protonated ions were dominated by a series of doubly charged y-ions and the amount of sequence information was dependent on the position of the histidine residue. In the peptides where the histidine was located towards the C-terminus of the peptide, a more extensive series of sequence specific product ions was observed. As the position of the histidine residue was moved towards the N-terminus of the peptide, systematically less sequence information was observed. The peptides were subsequently modified with diethylpyrocarbonate to manipulate the product ion spectra. Addition of the ethoxyformyl group to the N-terminus and histidine residue shifted the predominant charge state of the modified peptide to the doubly protonated form. These peptide ions fragmented efficiently, producing product ion spectra that contained more sequence information than could be obtained from the corresponding unmodified peptide.     

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.     

Snake venomics. Strategy and applications

Snake bites can be deadly, but the venoms also contain components of medical and biotechnological value. The proteomic characterization of snake venom proteomes, snake venomics, has thus a number of potential benefits for basic research, clinical diagnosis, and development of new research tools and drugs of potential clinical use. Snake venomics is also relevant for a deep understanding of the evolution and the biological effects of the venoms, and to generate immunization protocols to elicit toxin-specific antibodies with greater specificity and effectiveness than conventional systems. Our snake venomics approach starts with the fractionation of the crude venom by reverse-phase HPLC, followed by the initial characterization of each protein fraction by combination of N-terminal sequencing, SDS-PAGE, and mass spectrometric determination of the molecular masses and the cysteine (SH and S--S) content. Protein fractions showing a single electrophoretic band, molecular mass, and N-terminal sequence can be straightforwardly assigned by BLAST analysis to a known protein family. On the other hand, protein fractions showing heterogeneous or blocked N-termini are analyzed by SDS-PAGE and the bands of interest subjected to automated reduction, carbamidomethylation, and in-gel tryptic digestion. The resulting tryptic peptides are then analyzed by MALDI-TOF mass fingerprinting followed by amino acid sequence determination of selected doubly and triply charged peptide ions by collision-induced dissociation tandem mass spectrometry. The combined strategy allows us to assign unambiguously all the isolated venom toxins representing over 0.05% of the total venom proteins to known protein families. Protocols and applications of snake venomics are reviewed and discussed.     

Multiply charged cluster ions of Ar,Kr, Xe,N2, O2, CO2, SO2 and NH3: Production mechanism,appearance size and appearance energy

Clusters of Ar, Kr, Xe, N₂, O₂, CO₂, SO₂ and NH₃ formed by supersonic nozzle expansion have been studied by electron impact ionization mass spectrometry (up to 15000 amu). Besides mass spectra of singly charged ions showing the characteristic anomalous distributions, we have in particular investigated the properties of multiply charged cluster ions. Critical appearance sizes of doubly and triply charged cluster ions, n₂ and n₃ respectively, found in the present study confirm recent theoretical predictions about n₃/n₂ and their dependence on the properties of the cluster constituents. The appearance energies of multiply charged cluster ions determined are shifted way below the appearance energies of the respective monomer ions. These huge red shifts together with the observed linear threshold laws and large maximum ionization efficiencies indicate that multiply charged cluster ions are produced by sequential single ionization events of one incoming electron at different cluster sites. Furthermore, we have also obtained for the first time clear evidence that (for electron energies above the appearance energy of doubly charged ions) an appreciable amount of singly charged (also fragment) ions is produced via Coulomb explosion of unstable doubly charged ions in the ion source.     

Double photoionization of C(60) and C(70) in the valence region

Photoion yields from gaseous fullerenes, C(60) and C(70), for production of singly and doubly charged ions are measured by mass spectrometry combined with tunable synchrotron radiation at hnu=25-150 eV. Since the signal of triply or highly charged ions is very weak, the total photoionization yield curve can be estimated from the sum of the yields of the singly and doubly charged ions. A distinct feature appears in the resultant curve of C(60) which is absent in the calculated total photoabsorption cross section previously reported. This difference is attributed to C(60) (2+) ions chiefly produced by spectator Auger ionization of the shape resonance states followed by tunneling of the trapped electron or by cascade Auger ionization. Ratios between the yields of doubly and singly charged ions for C(60) and C(70) are larger than unity at hnu>50 eV. These ratios are quite different from those reported in the experiments using electron impact ionization.     

Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization

Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions.

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Radical <Emphasis Type="BoldItalic">a</Emphasis>-Ions in Electron Capture Dissociation: On the Origin of Species

Radical a* ions appear in electron capture dissociation mass spectra sporadically, but sometimes with high intensity. Mechanistically, radical a ions are hypothesized to arise due to thermodynamically disadvantaged charge solvation on the backbone nitrogen (instead of carbonyl), which upon neutralization produces a hypervalent group instantly fragmenting into a radical b* and conventional y' ion. The former species is unstable and, after releasing a CO molecule, decays to an a* ion. Here we validate this scenario by direct observation of the complementarity of a* and y' ions by interrogation of an ECD MS/MS database of >10,000 doubly and >5,000 triply charged tryptic peptides. Intriguingly, the most abundant a*/y' pairs are found to come from the cleavage of the same backbone link as the most abundant c' and z* complementary ions. This result gives strong support to the “local” N-Cα bond cleavage mechanism, in which the dissociation occurs at the site of charge solvation. However, a second strong peak is observed in the c'/z* fragment distribution four residues away from the a*/y' cleavage, which supports the indirect N-Cα bond cleavage mechanism. The size distribution of a ions from doubly (but not triply!) charged precursors shows deficit of a3 ions, and possibly a6 ions.     

Doubly charged ion mass spectra: V—Oxygen containing hydrocarbons

Doubly charged ion mass spectra were obtained for 46 low molecular weight oxygen containing compounds. A double focusing Hitachi RMU-7L mass spectrometer, operated at 3.2 kV accelerating voltage, was used to measure spectra for aliphatic alcohol, ketone, ether, aldehyde, ester and acid molecules, as well as several aromatic oxygen containing compounds. In general, the spectra were dominated by fragment ions which resulted from extensive H loss and C? C bond rupture as well as O elimination from the doubly charged molecular ions. Total product ion intensities from doubly charged ion spectra of aliphatic oxygen containing compounds were approximately 1% of the corresponding total ion intensity in the benzene doubly charged ion spectrum. Appearance energies for forming prominent doubly charged molecular and fragment ions were determined. Measured values ranged from 26 to 45 eV. A geometry optimized quantum mechanical SCF treatment was used to compute the energies, charge densities and structures for several of these oxygen containing doubly charged ions.     

Doubly charged ion mass spectra. 7—acetylenes

Doubly charged ion mass spectra of 20 aliphatic and 3 aromatic acetylenic compounds have been measured using a double focusing Hitachi RMU-7L mass spectrometer. Spectra were obtained using 100 eV ionizing electron energy and 3.2 kV ion accelerating voltage. In general, the spectra of aliphatic type acetylenic compounds were dominated by fragment ions formed by extensive H loss from doubly charged molecular ions. Intense molecular ions were observed in the doubly charged ion spectra of phenyl-substituted acetylenes. Total product ion intensities for doubly charged ion spectra of acetylenic compounds were found to be smaller, in general, than the total product ion intensity observed in the benzene doubly charged ion mass spectrum. Measured appearance energies of intense product ions ranged from 24 to 47 eV. A geometry optimized quantum mechanical self-consistent field molecular orbital treatment was employed to compute energies and structural parameters of prominent ions in the doubly charged ion mass spectra of acetylenic compounds.