Electron capture and collisionally activated dissociation mass spectrometry of doubly charged hyperbranched polyesteramides

Electron capture dissociation (ECD) of doubly protonated hyperbranched polyesteramide oligomers (1100-1900 Da) was examined and compared with the structural information obtained by low energy collisionally activated dissociation (CAD). Both the ester and amide bonds of the protonated species were cleaved easily upon ECD with the formation of odd electron (OE(.+)) or even electron (EE(+)) fragment ions. Several mechanistic schemes are proposed that describe the complex ECD fragmentation behavior of the multiply charged oligomers. In contrast to studies of biomolecules, the present results indicate that consecutive cleavages induced by intramolecular H-shifts are significant for ECD and of less importance for low energy CAD. The capture of an electron by the ionized species results in fragmentation associated with a redistribution of the excess internal energy over the products and the subsequent bond cleavage. Low energy, multiple collision CAD is found to be a more selective dissociation method than ECD in view of the observation that only amide bonds are cleaved for most of the hyperbranched polymers examined with CAD in this study. ECD appears not to provide complementary structural information compared to CAD in the study of hyperbranched polymers, even though a significantly more complex ECD fragmentation behavior is observed. ECD is shown to be of use for the structural characterization of large oligomers that may not dissociate upon low energy CAD. This is a direct result of the fact that ECD produces ionized hyperbranched oligomers with a relatively high internal energy.     

Electron capture induced dissociation of nucleotide anions in water nanodroplets

We have studied the outcome of collisions between the hydrated nucleotide anion adenosine 5'-monophosphate (AMP) and sodium. Electron capture leads to hydrogen loss as well as water evaporation regardless of the initial number m of water molecules attached to the parent ion (m< or =16). The yield of dianions with microsecond lifetimes increases strongly with m, which is explained from dielectric screening of the two charges by the water nanodroplet. For comparison, collision induced dissociation results in water losses with no or very little damage of the AMP molecule itself.     

Electron capture dissociation of complexes of diacylglycerophosphocholine and divalent metal ions: Competition between charge reduction and radical induced phospholipid fragmentation

Divalent metal complexes of phosphocholines, [Metal(II)(L)(n)](2+) (where Metal=Cu(2+), Co(2+), Mg(2+), and Ca(2+), L=1,2-dihexanoyl-sn-glycero-3-phosphocholine [6:0/6:0GPCho] and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [16:0/18:1GPCho] and n=2-5), were formed upon electrospray ionization mass spectrometry (ESI/MS) of 8 mM solution of phosphocholine (L) with 4 mM metal salt (Metal). The electron capture dissociation (ECD) reactions of these [Metal(II)(L)(n)](2+) complexes were examined via Fourier-transform ion-cyclotron resonance mass spectrometry. A rich and complex chemistry was observed, including charge reduction and fragmentation involving losses of a methyl radical, trimethylamine, and the acyl chains. The predominant reaction channel was dependent on the size (n) of the complex, the metal and ligand used, and the size of the acyl chain. Thus charge reduction dominates the ECD spectra of the larger phosphocholine, 16:0/18:1GPCho, but is largely absent in the smaller 6:0/6:0GPCho. For complexes of 16:0/18:1GPCho, n=4-5, fragmentation from the head group mainly occurs via loss of the methyl radical and trimethylamine. At n=3, the relative abundance of fragments due to loss of acyl chain radicals increases. The abundances of ions arising from these radical losses increase further for the n=2 complexes, thereby providing information on the composition and position of the 16:0 and 18:1 acyl groups. Thus ECD of metal complexes provides structurally useful information on the phosphocholine, including the nature of the head group, the acyl chains, and the positions of the acyl chains.     

Electron‐induced dissociation of doubly protonated betaine clusters: controlling fragmentation chemistry through electron energy

The [M₂₁+2H]²⁺ cluster of the zwitterion betaine, M = (CH₃)₃NCH₂CO₂, formed via electrospray ionisation (ESI), has been allowed to interact with electrons with energies ranging from >0 to 50 eV in a Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometer. The types of gas‐phase electron‐induced dissociation (EID) reactions observed are dependent on the energy of the electrons. In the low‐energy region up to 10 eV, electrons are mainly captured, forming the charge‐reduced species, {[M₂₁+2H]^{+ .} }*, in an excited state, which stabilises via the ejection of an H atom and one or more neutral betaines. In the higher energy region, above 12 eV, a Coulomb explosion of the multiply charged clusters is observed in highly asymmetric fission with singly charged fragments carrying away more than 70% of the parent mass. Neutral betaine evaporation is also observed in this energy region. In addition, a series of singly charged fragments appears which arise from C? X bond cleavage reactions, including decarboxylation and CH₃ group transfer. These latter reactions may arise from access of electronic excited states of the precursor ions. Copyright © 2009 John Wiley & Sons, Ltd.     

The structure and dissociation pathways of protonated methanol: An ab initio molecular orbital study

Ab initio molecular orbital calculations with moderately large polarization basis sets and including valence-electron correlation have been used to examine the structure and dissociation mechanisms of protonated methanol [CH₃OH₂]⁺. Stable isomers and transition structures have been characterized using gradient techniques. Protonated methanol is found to be the only stable isomer in the [CH₅O]⁺ potential surface. There is no evidence for a tightly-bound complex, [HOCH₂]⁺…?H₂, analogous to the preferred structure [CH₃]⁺…?H₂ of [CH₅]⁺. Protonated methanol is found to possess a pyramidal arrangement of bonds at the oxygen atom with a barrier to inversion of 8kJ mol^?1. The lowest energy fragmentation pathways are dissociation into methyl cation and water (predicted to require 284 kJ mol^?1 with zero reverse activation energy) and loss of molecular hydrogen (endothermic by 138 kJ mol^?1 but with a reverse activation barrier of 149 kJ mol^?1). The results offer a possible explanation as to why production of [CH₂OH]⁺ from the reaction of methyl cation with water is not observed. Other dissociation processes examined include loss of a hydrogen atom to yield the methylenoxonium radical cation or methanol radical cation (requiring 441 and 490 kJ mol^?1, respectively) and loss of a proton to yield neutral methanol (requiring 784 kJ mol^?1).     

Electron capture dissociation and infrared multiphoton dissociation of oligodeoxynucleotide dications

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

Electron transfer dissociation of doubly sodiated glycerophosphocholine lipids

The ability to generate gaseous doubly charged cations of glycerophosphocholine (GPC) lipids via electrospray ionization has made possible the evaluation of electron-transfer dissociation (ETD) for their structural characterization. Doubly sodiated GPC cations have been reacted with azobenzene radical anions in a linear ion trap mass spectrometer. The ion/ion reactions proceed through sodium transfer, electron-transfer, and complex formation. Electron-transfer reactions are shown to give rise to cleavage at each ester linkage with the subsequent loss of a neutral quaternary nitrogen moiety. Electron-transfer without dissociation produces [M + 2Na](+.) radical cations, which undergo collision-induced dissociation (CID) to give products that arise from bond cleavage of each fatty acid chain. The CID of the complex ions yields products similar to those produced directly from the electron-transfer reactions of doubly sodiated GPC, although with different relative abundances. These findings indicate that the analysis of GPC lipids by ETD in conjunction with CID can provide some structural information, such as the number of carbons, degree of unsaturation for each fatty acid substituent, and the positions of the fatty acid substituents; some information about the location of the double bonds may be present in low intensity CID product ions.     

A study of the electron capture induced decompositions and charge separation reactions of the doubly charged isomeric ions of the methylpyridines and aniline

The doubly charged isomeric ions [C₆H₇N]²⁺ formed from 2-, 3- and 4-methylpyridine and aniline were investigated via their unimolecular charge separation reactions and by electron capture induced decompositions (ECID). The ECID spectra were compared with the collision induced decomposition (CID) spectra of the singly charged ions in an attempt to investigate the structure of the doubly charged ions. The four isomers could be unambiguously identified by their unimolecular charge separations. These differences were greater than in the mass spectra, ECID spectra or CID spectra of singly charged ions.     

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.     

Letter: intercluster chemistry of protonated and sodiated betaine dimers upon collision induced dissociation and electron induced dissociation

The collision induced dissociation and electron induced dissociation spectra of the [2M + H](+) and [2M + Na](+) clusters of the zwitterionic amino acid, betaine (M), have been examined in a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. Intercluster reactions are observed in the collision induced dissociation spectra of [2M + H](+) and [2M + Na](+) and in the electron induced dissociation spectrum of [2M + H](+).     

Generation and dissociation pathways of singly and doubly protonated bisguanidines in the gas phase

Para-bisguanidinyl benzene 1 and its N-permethylated derivative 2 are both sufficiently strong bases to afford not only the monocations [1+H]+ and [2+H]+, but also the doubly protonated ions, [1+2H]2+ and [2+2H]2+, in the gas phase. The title ions generated via electrospray ionization are probed by collision-induced dissociation experiments which inter alia reveal that the dicationic species [1+2H]2+ and [2+2H]2+ can even undergo fragmentation reactions with maintenance of the 2-fold charge. Complementary results from density functional theory predict PAs above 1000 kJ mol(-1) for the neutral compounds, i.e., PA(1) = 1025 kJ mol(-1) and PA(2) = 1067 kJ mol(-1). Due to the stabilization of the positive charge in the guanidinium ions and the para-phenylene spacer separating the basic sites, even the monocations bear sizable proton affinities, i.e., PA([1+H]+) = 740 kJ mol(-1) and PA([2+H]+) = 816 kJ mol(-1).     

The effect of fixed charge modifications on electron capture dissociation

Electron capture dissociation (ECD) studies of two modified amyloid beta peptides (20-29 and 25-35) were performed to investigate the role of H* radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H* generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z* ions were still observed in their ECD spectra. Thus, although H* transfer may be important for the NC(alpha) bond cleavage, there also exist other pathways, which would require a radical migration via H* abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides.     

Electron capture dissociation of singly and multiply phosphorylated peptides

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

苯二胺双电荷离子的电荷分离和电子捕获诱导解离谱研究

本工作用电荷分离(CS)谱和电子捕获诱导解离(ECID)谱研究了三种苯二胺异构体的双电荷分子离子在气相中的结构和反应。三种异构体双电荷离子的电荷分离反应和电子捕获诱导解离反应有相同的反应通道。通过测量不同反应通道的动能释放, 推测出了双电荷离子解反应过渡状态的结构。另外, 三种异构体可用ECID谱来区分。     

Electron capture versus energetic dissociation of protein ions

The identity of neighboring amino acids has little influence on the dissociation of multiply protonated proteins by electron capture dissociation. As exceptions, no cleavage occurs on the N-terminal side of Pro, and little on either side of Cys, whereas the C-terminal side of Trp is heavily favored. The neighboring amino acids have a far greater effect on energetic dissociation, making the combined methods promising for the de novo sequencing of proteins.     

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.     

Electron capture dissociation of weakly bound polypeptide polycationic complexes

We have previously reported that, in electron capture dissociation (ECD), rupture of strong intramolecular bonds in weakly bound supramolecular aggregates can proceed without dissociation of weak intermolecular bonds. This is now illustrated on a series of non-specific peptide-peptide dimers as well as specific complexes of modified glycopeptide antibiotics with their target peptide. The weak nature of bonding is substantiated by blackbody infrared dissociation, low-energy collisional excitation and force-field simulations. The results are consistent with a non-ergodic ECD cleavage mechanism.     

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains.     

Collision‐induced dissociation processes of protonated benzoic acid and related compounds: competitive generation of protonated carbon dioxide or protonated benzene

Upon activation in the gas phase, protonated benzoic acid (m/z 123) undergoes fragmentation by several mechanisms. In addition to the predictable water loss followed by a CO loss, the m/z 123 ion more intriguingly eliminates a molecule of benzene to generate protonated carbon dioxide (H ‐ O⁺ ═ C ≡ O , m/z 45), or a molecule of carbon dioxide to yield protonated benzene (m/z 79). Experimental evidence shows that the incipient proton ambulates during the fragmentation processes. For the CO₂ or benzene loss, protonated benzoic acid transfers the charge‐imparting proton initially to the ortho position and then to the ipso position to generate a transient species which dissociates to form an ion‐neutral complex between benzene and protonated CO₂. The formation of the m/z 45 ion is not a phenomenon unique to benzoic acid: spectra from protonated isophthalic acid, terephthalic acid, trans‐cinnamic acid and some aliphatic acids also displayed a peak for m/z 45. However, the m/z 45 peak is structurally diagnostic only for certain benzene polycarboxylic acids because the spectra of compounds with two carboxyl groups on adjacent ring carbons do not produce a peak at m/z 45. For the m/z 79 ion to be formed, an intramolecular reaction should take place in which protonated CO₂ within the ion‐neutral complex acts as the attacking electrophile to transfer a proton to benzene. Copyright © 2017 John Wiley & Sons, Ltd.     

Electron capture dissociation distinguishes a single D-amino acid in a protein and probes the tertiary structure

First results are reported on the application of ECD in analysis of 2+ and 3+ ions of stereoisomers of Trp-cage (NLYIQWLKDGGPSSGRPPPS), the smallest and fastest-folding protein, which exhibits a tightly folded tertiary structure in solution. The chiral recognition based on the ratios of the abundances of z(18) and z(19) fragments in ECD of 2+ ions was excellent even for a single amino acid (Tyr) D-substitution (R(chiral) = 8.6). The chiral effect decreased with an increase of temperature at the electrospray ion source, as well as at a higher degree of ionization, 3+ ions (R(chiral) = 1.5). A general approach is suggested for charge localization in n+ ions by analysis of ECD mass spectra of (n + 1)+ ions. Application of this approach to 3+ Trp-cage ions revealed the protonation probability order in 2+ ions: Arg(16) > Gln(5) > approximately N-terminus. The ECD results for native form of the 2+ ions favor the preservation of the solution-phase tertiary structure, and chiral recognition through the interaction between the charges and the neutral bond network. Conversely, ECD of 3+ ions supports the dominance of ionic hydrogen bonding which determines a different gas-phase structure than found in solution. Vibrational activation of 2+ ions indicated greater stability of the native form, but the fragmentation patterns did not provide stereoisomer differentiation, thus underlying the special position of ECD among other MS/MS fragmentation techniques. Further ECD studies should yield more structural information as well as quantitative single-amino acid D/L content measurements in proteins.