Electron transfer dissociation of peptide anions

Ion/ion reactions of multiply deprotonated peptide anions with xenon radical cations result in electron abstraction to generate charge-reduced peptide anions containing a free-radical site. Peptide backbone cleavage then occurs by hydrogen radical abstraction from a backbone amide N to facilitate cleavage of the adjacent C-C bond, thereby producing a- and x-type product ions. Introduction of free-radical sites to multiply charged peptides allows access to new fragmentation pathways that are otherwise too costly (e. g., lowers activation energies). Further, ion/ion chemistry, namely electron transfer reactions, presents a rapid and efficient means of generating odd-electron multiply charged peptides; these reactions can be used for studying gas-phase chemistries and for peptide sequence analysis.     

Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z ^• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.     

Electron transfer dissociation versus collisionally activated dissociation of cationized biodegradable polyesters

Biodegradable polyesters were ionized by electrospray ionization and characterized by tandem mass spectrometry using collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) as activation methods. The compounds studied include one homopolymer, polylactide and two copolymers, poly(ethylene adipate) and poly(butylene adipate). CAD of [M+2Na]²⁺ ions from these polyesters proceeds via charge‐remote 1,5‐H rearrangements over the ester groups, leading to cleavages at the (CO)O–alkyl bonds. ETD of the same precursor ions creates a radical anion at the site of electron attachment, which fragments by radical‐induced cleavage of the (CO)O–alkyl bonds and by intramolecular nucleophilic substitution at the (CO)–O bonds. In contrast to CAD, ETD produces fragments in one charge state only and does not cause consecutive fragmentations, which simplifies spectral interpretation and permits conclusive identification of the correct end groups. The radical‐site reactions occurring during ETD are very similar with those reported for ETD of protonated peptides. Unlike multiply protonated species, multiply sodiated precursors form ion pairs (salt bridges) after electron transfer, thereby promoting dissociations via nucleophilic displacement in addition to the radical‐site dissociations typical in ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

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 transfer dissociation of amide nitrogen methylated polypeptide cations

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process.     

Electron transfer dissociation of dipositive uranyl and plutonyl coordination complexes

Reported here is a comparison of electron transfer dissociation (ETD) and collision‐induced dissociation (CID) of solvent‐coordinated dipositive uranyl and plutonyl ions generated by electrospray ionization. Fundamental differences between the ETD and CID processes are apparent, as are differences between the intrinsic chemistries of uranyl and plutonyl. Reduction of both charge and oxidation state, which is inherent in ETD activation of [An^VIO₂(CH₃COCH₃)₄]²⁺, [An^VIO₂(CH₃CN)₄]², [U^VIO₂(CH₃COCH₃)₅]²⁺ and [U^VIO₂(CH₃CN)₅]²⁺ (An = U or Pu), is accompanied by ligand loss. Resulting low‐coordinate uranyl(V) complexes add O₂, whereas plutonyl(V) complexes do not. In contrast, CID of the same complexes generates predominantly doubly‐charged products through loss of coordinating ligands. Singly‐charged CID products of [U^VIO₂(CH₃COCH₃)_4,5]²⁺, [U^VIO₂(CH₃CN)_4,5]²⁺ and [Pu^VIO₂(CH₃CN)₄]²⁺ retain the hexavalent metal oxidation state with the addition of hydroxide or acetone enolate anion ligands. However, CID of [Pu^VIO₂(CH₃COCH₃)₄]²⁺ generates monopositive plutonyl(V) complexes, reflecting relatively more facile reduction of Pu^VI to Pu^V. Copyright © 2011 John Wiley & Sons, Ltd.     

Electron capture induced dissociation of doubly protonated pentapeptides: dependence on molecular structure and charge separation

We have studied electron capture induced dissociation of a set of doubly protonated pentapeptides, all composed of one lysine (K) and either four glycine (G) or four alanine (A) residues, as a function of the sequence of these building blocks. Thereby the separation of the two charges, sequestered on the N-terminal amino group and the lysine side chain, is varied. The characteristic cleavage of N-C(α) bonds is observed for all peptides over the whole backbone length, with the charge carrying fragments always containing K. The resulting fragmentation patterns are very similar if G is replaced by A. In the case of [XKXXX+2H](2+) (X=A or G), a distinct feature is observed in the distribution of backbone cleavage fragments and the probability for ammonia loss is drastically reduced. This may be due to an isomer with an amide oxygen as protonation site giving rise to the observed increase in breakage at a specific site in the molecule. For the other peptides, a correlation with the distance between amide oxygen and the charge at the lysine side chain has been found. This may be an indication that it is only the contribution from this site to the charge stabilization of the amide π(*) orbitals which determines relative fragment intensities. For comparison, complexes with two crown ether molecules have been studied as well. The crown ether provides a shielding of the charge and prevents the peptide from folding and internal hydrogen bonding, which leads to a more uniform fragmentation behavior.     

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](+).     

Electron attachment step in electron capture dissociation (ECD) and electron transfer dissociation (ETD)

We have made use of classical dynamics trajectory simultions and ab initio electronic structure calculations to estimate the cross sections with which electrons are attached (in electron capture dissociation (ECD)) or transferred (in electron transfer dissociation (ETD)) to a model system that contained both an S-S bond that is cleaved and a -NH(3)(+) positively charged site. We used a Landau-Zener-Stueckelberg curve-crossing approximation to estimate the ETD rates for electron transfer from a CH(3)(-) anion to the -NH(3)(+) Rydberg orbital or the S-S sigma* orbital. We draw conclusions about ECD from our ETD results and from known experimental electron-attachment cross sections for cations and sigma-bonds. We predict the cross section for ETD at the positive site of our model compound to be an order of magnitude larger than that for transfer to the Coulomb-stabilized S-S bond site. We also predict that, in ECD, the cross section for electron capture at the positive site will be up to 3 orders of magnitude larger than that for capture at the S-S bond site. These results seem to suggest that attachment to such positive sites should dominate in producing S-S bond cleavage in our compound. However, we also note that cleavage induced by capture at the positive site will be diminished by an amount that is related to the distance from the positive site to the S-S bond. This dimunition can render cleavage through Coulomb-assisted S-S sigma* attachment competitive for our model compound. Implications for ECD and ETD of peptides and proteins in which SS or N-C(alpha) bonds are cleaved are also discussed, and we explain that such events are most likely susceptible to Coulomb-assisted attachment, because the S-S sigma* and C=O pi* orbitals are the lowest-lying antibonding orbitals in most peptides and proteins.     

Analysis of oxidized glycerophosphocholine lipids using electrospray ionization mass spectrometry and microderivatization techniques

Oxidized low-density lipoprotein (LDL) is thought to play an important role in atherogenesis and cardiovascular disease in humans. Oxidized LDL is a complex mixture of many oxidized species, including numerous oxidized glycerophospholipids. Electrospray ionization and tandem mass spectrometry as well as microchemical derivatization of high-performance liquid chromatographically purified fractions derived from oxidized LDL were investigated as means to determine the structure of individual components present in oxidized LDL. One major oxidized phosphocholine lipid had an [M + H](+) ion at m/z 650. Derivatization to the trimethylsilyl ether and methoxime caused shifts in mass which, along with negative ion collision-induced dissociation mass spectra, were consistent with the presence of three species, 1-palmitoyl-2-(9-oxononanoyl)glycerophosphocholine and two isomeric 1-octadecanoyl-2-(hydroxyheptenoyl)glycerophosphocholines. These species were chemically synthesized. Trimethylsilylation of free hydroxyl groups increased the mass of the phospholipid acyl chains containing hydroxyl groups by 72 u. Conversion of carbonyl groups to the methoxylamine derivative increased the mass by 29 u. Ozonolysis of those products which contained double bonds proved to be a facile technique to determine the position and number of double bonds present. The use of these techniques was illustrated in the structural characterization of one major component (m/z 650, positive ions) in oxidized LDL as 1-octadecanoyl-2-(7-hydroxyhepta-5-enoyl)glycerophosphocholi ne. A possible mechanism for the formation of this unique chain-shortened glycerophospholipid is proposed.     

Electron transfer dissociation of N-glycopeptides: loss of the entire N-glycosylated asparagine side chain

The recently introduced electron transfer dissociation (ETD) technique opens new possibilities for the structural characterization of glycoproteins at the glycopeptide level. In this report, we investigate the ETD mass spectra of tryptic N-glycopeptides of the model glycoprotein horseradish peroxidase (HRP). Multiply protonated N-glycopeptides obtained by electrospray ionization were subjected to ETD. Fragment ions obtained by ETD were further analyzed by collision-induced dissociation (CID) (MS(3)) for their unambiguous structural assignment. The following fragmentation features were revealed: (1) c- and z-type peptide backbone cleavages were observed with retention of the intact glycan moiety revealing peptide sequence, glycan attachment site, and glycan mass; (2) to a lesser extent, glycosidic bond cleavages were registered with retention of the intact peptide sequence; and (3) a range of amino acid side chain losses did occur. Remarkably, the loss of the complete N-glycosylated asparagine side chain was observed. This loss of the glycan-modified side chain helps with the structural characterization of glycopeptides by allowing the facile deduction and verification of the glycan mass and the nature of the amino acid residue at the glycan attachment site. Importantly, informative ETD spectra were obtained in this study by reversed-phase nano-liquid chromatography (LC) coupled online to a radio-frequency (rf) quadrupole ion trap (QIT) mass spectrometer with alternating acquisition of CID and ETD mass spectra from an automatically selected set of precursors (data-dependent mode). Thus, our study brings nano-LC/QIT-MS(n) with CID and ETD to the fore as a powerful technique for glycoproteomics at the glycopeptide level.     

Determination of the glycosylation site in flavonoid mono-O-glycosides by collision-induced dissociation of electrospray-generated deprotonated and sodiated molecules

The influence of the glycosylation site on the fragmentation behavior of 18 flavonoid glycoside standards was studied using positive and negative electrospray ionization mass spectrometry in combination with collision-induced dissociation and tandem mass spectrometry. The glycosylation position is shown to affect the relative abundance of the radical aglycone ions that can be observed in the [M-H]- collision-induced dissociation spectra. In particular, the radical aglycone ions are very abundant for deprotonated flavonol 3-O-glycosides. Collisional activation of the radical aglycone ions produced from positional isomers revealed minor differences: m,nB0- product ions are pronounced for 7-O-glycosides, whereas m,nA0- product ions are relatively more abundant for 4'-O-glycosides. In addition, the ratio between the radical aglycone and the regular aglycone ions in the [M+Na]+ high-energy collision-induced dissociation spectra gives an indication about the glycosylation site. This ion ratio allows the differentiation between flavonoid 3-O- and 7-O-glycosides or can be useful in the comparison of unknown compounds with standards. Unambiguous differentiation between O-glycosylation at the common positions of flavonoid O-glycosides, i.e. the 3-, 4'- and 7-positions, is achieved by collisional activation of sodiated molecules at high collision energy. The presence of a B-ring product ion containing the sugar residue indicates 4'-O-glycosylation, whereas the loss of the B-ring part from the aglycone product ion is characteristic of 3-O-glycosylation and the loss of the B-ring part from both the [M+Na]+ precursor ion and the aglycone product ion points to 7-O-glycosylation.     

Structure of sodiated polyglycines

The intrinsic folding of peptides about a sodium ion has been investigated in detail by using infrared multiple photon dissociation (IRMPD) spectroscopy and a combination of theoretical methods. IRMPD spectroscopy was carried out on sodiated polyglycines G(n)-Na(+) (n=2-8), in both the fingerprint and N-H/O-H stretching regions. Interplay between experimental and computational approaches (classical and quantum) enables us to decipher most structural details. The most stable structures of the small peptides up to G(6)-Na(+) maximize metal-peptide interactions with all peptidic C=O groups bound to sodium. In addition, direct interactions between peptide termini are possible for G(6)-Na(+) and larger polyglycines. The increased flexibility of larger peptides leads to more complex folding and internal peptide structuration through γ or β turns. A structural transition is found to occur between G(6)-Na(+) and G(7)-Na(+), leading to a structure with sodium coordination that becomes tri-dimensional for the latter. This transition was confirmed by H/D exchange experiments on G(n)-Na(+) (n=3-8). The most favorable hydrogen-bonding pattern in G(8)-Na(+) involves direct interactions between the peptide termini and opens the way to salt-bridge formation; however, there is only good agreement between experimental and computational data over the entire spectral range for the charge solvation isomer.     

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 detachment dissociation of glycosaminoglycan tetrasaccharides

The first application of electron detachment dissociation (EDD) to carbohydrates is presented. The structural characterization of glycosaminoglycan (GAG) oligosaccharides by mass spectrometry is a longstanding problem because of the lability of these acidic, polysulfated carbohydrates. Doubly-charged negative ions of four GAG tetrasaccharides are examined by EDD, collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD). EDD is found to produce information-rich mass spectra with both cross ring and glycosidic cleavage product ions. In contrast, most of the product ions produced by CAD and IRMPD result from glycosidic cleavage. EDD shows great potential as a tool for locating the sites of sulfation and other modifications in glycosaminoglycan oligosaccharides.     

Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry

Electrospray ionization (ESI) tandem mass spectrometry (MS) has simplified analysis of phospholipid mixtures, and, in negative ion mode, permits structural identification of picomole amounts of phospholipid species. Collisionally activated dissociation (CAD) of phospholipid anions yields negative ion tandem mass spectra that contain fragment ions representing the fatty acid substituents as carboxylate anions. Glycerophosphocholine (GPC) lipids contain a quaternary nitrogen moiety and more readily form cationic adducts than anionic species, and positive ion tandem mass spectra of protonated GPC species contain no abundant ions that identify fatty acid substituents. We report here that lithiated adducts of GPC species are readily formed by adding lithium hydroxide to the solution in which phospholipid mixtures are infused into the ESI source. CAD of [MLi⁺] ions of GPC species yields tandem mass spectra that contain prominent ions representing losses of the fatty acid substituents. These ions and their relative abundances can be used to assign the identities and positions of the fatty acid substituents of GPC species. Tandem mass spectrometric scans monitoring neutral losses of the head-group or of fatty acid substituents from lithiated adducts can be used to identify GPC species in tissue phospholipid mixtures. Similar scans monitoring parents of specific product ions can also be used to identify the fatty acid substituents of GPC species, and this facilitates identification of distinct isobaric contributors to ions observed in the ESI/MS total ion current.     

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.     

Electron detachment dissociation of dermatan sulfate oligosaccharides

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

Electron transfer dynamics

This paper surveys the current ‘state-of-art’ of the theoretical understanding of electron transfer dynamics in donor-acceptor systems, which provide the conceptual and technical basis for solar energy conversion via optical and optoelectronic molecular devices and for the primary charge separation in photo-synthesis.