High-energy Electron Transfer Dissociation Research Articles

Study of Ion Dynamics by Electron Transfer Dissociation: Alkali Metals as Targets.

High energy collision processes for singly charged positive ions using an alkali metal target are confirmed, as a charge inversion mass spectrometry, to occur by electron transfers in successive collisions and the dissociation processes involve the formation of energy-selected neutral species from near-resonant neutralization with alkali metal targets. A doubly charged thermometer molecule was made to collide with alkali metal targets to give singly and doubly charged positive ions. The internal energy resulting from the electron transfer with the alkali metal target was very narrow and centered at a particular energy. This narrow internal energy distribution can be attributed to electron transfer by Landau-Zener potential crossing between the precursor ion and an alkali metal atom, and the coulombic repulsion between singly charged ions in the exit channel. A large cross section of more than 10-14 cm2 was estimated for high-energy electron transfer dissociation (HE-ETD). Doubly protonated phosphorylated peptides obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions. Whereas doubly charged fragment ions resulting from CAD were dominant in the case of the Xe target, singly charged fragment ions resulting from ETD were dominant with the Cs target. HE-ETD using the Cs target provided all of the z-type ions by N-Cα bond cleavage without the loss of the phosphate groups. The results demonstrate that HE-ETD with an alkali metal target allowed the position of phosphorylation and the amino acid sequence of peptides with post translational modifications (PTM) to be determined.

High-energy electron transfer dissociation of protonated amino acids

High-energy electron transfer dissociation (HE-ETD) of singly protonated amino acids was investigated using charge inversion mass spectrometry with alkali metal targets in which dissociations of charge-reduced neutrals were induced from the excited neutral intermediates. The dominant trapezoidal peaks of the 17 u loss in the charge inversion spectra of the protonated aliphatic amino acids were assigned to NH3 loss caused by NCα bond cleavage from hypervalent ammonium radical intermediates via a repulsive state; this assignment was based on the peak shape and kinetic energy release (KER) values. The peak of the non-dissociated ion observed in the proline spectra was explained by the same bond cleavage. The triangular peaks of the 2 u loss, which were dominantly observed in all of the spectra, were assigned to H2 elimination, which involved H atom migration in the neutral radical. Neutral intermediates that induced the H2 loss were estimated to have a lower internal energy than that of the NH3 loss by comparing the charge inversion spectra of the different target atoms. Differences in the isobaric ions between leucine and isoleucine were found in the bond dissociation of CβCγ in their side chains following the NH3 loss and in the intensity of the NH3 loss relative to that of the H2 loss. The charge inversion spectra of the protonated amino acids, with side chains that are subject to post-translational modification, provided intense peaks resulting from the consecutive and competitive loss of neutral molecules following the NH3 loss and H2 loss. Along with these losses, side chain losses caused by H atom migration were also observed as sharp peaks with KER values smaller than that of the consecutive loss.

Dissociation mechanisms of excited CH3X (X = Cl, Br, and I) formed via high-energy electron transfer using alkali metal targets

High-energy electron transfer dissociation (HE-ETD) on collisions with alkali metal targets (Cs, K, and Na) was investigated for CH(3)X(+) (X = Cl, Br, and I) ions by a charge inversion mass spectrometry. Relative peak intensities of the negative ions formed via HE-ETD strongly depend on the precursor ions and the target alkali metals. The dependency is explained by the exothermicities of the respective dissociation processes. Peak shapes of the negative ions, especially of the X(-) ions, which comprise a triangle and a trapezoid, also strongly depend on the precursor ions and the target alkali metals. The trapezoidal part of the I(-) peak observed with the Na target is more dominant and much broader than that with the Cs target. This dependence on the targets shows an inverse relation between the peak width and the available energy, which corresponds to the exothermicity assuming formation of fragment pair in their ground internal states. From a comparison of the kinetic energy release value calculated from the trapezoidal shape of I(-) with the available energy of the near-resonant level on the CH(3)I potential energy curve reported by ab initio calculations, the trapezoidal part is attributed to the dissociation to CH(3) + I((2)P(3/2)) via the repulsive (3)Q(1) state of CH(3)I, which is not dominant in the photo-dissociation of CH(3)I. The observation of trapezoid shape of the CH(2)I(-) peak with the Cs target indicates spontaneous dissociation via repulsive potential from the (3)R(2) Rydberg state, although the correlation between the (3)R(2) Rydberg state and relevant repulsive states has not been reported by any theoretical calculation.

High-Energy Electron Transfer Dissociation (HE-ETD) Using Alkali Metal Targets for Sequence Analysis of Post-Translational Peptides

Post-translational modifications (PTMs) of proteins are important in the activation, localization, and regulation of protein function in vivo. The usefulness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) in tandem mass spectrometry (MS/MS) using low-energy (LE) trap type mass spectrometer is associated with no loss of a labile PTM group regarding peptide and protein sequencing. The experimental results of high-energy (HE) collision induced dissociation (CID) using the Xe and Cs targets and LE-ETD were compared for doubly-phosphorylated peptides TGFLT(p)EY(p)VATR (1). Although HE-CID using the Xe target did not provide information on the amino acid sequence, HE-CID using the Cs target provided all the z-type ions without loss of the phosphate groups as a result of HE-ETD process, while LE-ETD using fluoranthene anion gave only z-type ions from z(5) to z(11). The difference in the results of HE-CID between the Xe and Cs targets demonstrated that HE-ETD process with the Cs target took place much more dominantly than collisional activation. The difference between HE-ETD using Cs targets and LE-ETD using the anion demonstrated that mass discrimination was much weaker in the high-energy process. HE-ETD was also applied to three other phosphopeptides YGGMHRQEX(p)VDC (2: X = S, 3: X = T, 4: X = Y). The HE-CID spectra of the doubly-protonated phosphopeptides (= [M + 2H](2+)) of 2, 3, and 4 using the Cs target showed a very similar feature that the c-type ions from c(7) to c(11) and the z-type ions from z(7) to z(11) were formed via N-C alpha bond cleavage without a loss of the phosphate group.

Letter: High-Energy Electron Transfer Dissociation Using a Tandem Time-of-Flight Mass Spectrometer with an Electrospray Ionization Ion Source

A tandem time-of-flight (TOF) mass spectrometer with an electrospray ionization (ESI) ion source (ESI-TOF/quadTOF) was enhanced to perform high-energy electron transfer dissociation (ETD) experiments. Specifically, the collision cell for rare gas targets of ESI-TOF/quadTOF was replaced with one for alkali metal targets. High-energy ETD spectra were obtained of doubly protonated polypeptides and a phosphopeptide using a Cs target. Peaks due to several c- and z-type fragment ions formed by the high-energy ETD process were clearly observed in all cases. These results demonstrate that high-energy ETD can be performed using ESI-TOF/quadTOF.

高エネルギー電子移動解離が可能なタンデム質量分析装置の開発

A JEOL JMS-HX110 double-focusing mass spectrometer was combined with the collision cell and spherical electric sector of a HITACHI M80-B instrument to perform tandem mass spectrometry experiments. A quadrupole lens doublet was mounted between the mass spectrometer and the collision cell to improve ion transmission into the collision cell. The detection system was also improved to expand the mass range of product ions. These improvements provided reliable spectra of high-energy electron transfer dissociation (HE-ETD) of peptides using an alkali metal target. The present results demonstrated that HE-ETD by a single collision with an alkali metal target could determine the amino acid sequence and phosphorylation site of peptides.

Differences between collisionally activated and electron‐transfer dissociations found for CH2X2(X = Cl, Br, and I) by using alkali‐metal targets

High-energy collisionally activated dissociation (HE-CAD) and high-energy electron- transfer dissociation (HE-ETD) on collisions with alkali-metal targets (Cs, K, and Na) were investigated for CH(2)X(2) (+) (X = Cl, Br, and I) ions by tandem mass spectrometry (MS/MS). In the HE-CAD spectra observed, peaks associated with CH(2)X(+) ions formed by a loss of a halogen atom are always predominant regardless of precursor ions and target metals. The observation of the predominant CH(2)X(+) ions is explained by the lowest energy levels of the fragments of CH(2)X(+) + X among the possible fragment energy levels and internal-energy distribution in HE-CAD. In the charge-inversion spectra, relative peak intensities of the negative ions formed by HE-ETD strongly depend on the precursor ions and the target metals. While the CHCl(2) (-) ion was predominant in the spectra of CH(2)Cl(2) (+) regardless of target species, the most intense peaks in those of CH(2)Br(2) (+) and CH(2)I(2) (+) were ascribed to either Br(-) or CH(2)Br(-) and either I(-) or I(2) (-), respectively, depending on the target metals. The dependence of the relative intensities of the fragment ions by HE-ETD on the precursor ions and target species are discussed on the basis of the energy levels of the neutral fragments and the narrow internal-energy distribution resulting from the near-resonant neutralization. It was demonstrated that HE-ETD using the alkali-metal targets provided rich information on the dissociation of the neutral species.

Study of the dissociation of a charge‐reduced phosphopeptide formed by electron transfer from an alkali metal target

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.