James W. Hager

Mass selective axial ion ejection from a linear quadrupole ion trap.

The electric fields responsible for mass-selective axial ejection (MSAE) of ions trapped in a linear quadrupole ion trap have been studied using a combination of analytic theory and computer modeling. Axial ejection occurs as a consequence of the trapped ions’ radial motion, which is characterized by extrema that are phase-synchronous with the local RF potential. As a result, the net axial electric field experienced by ions in the fringe region, over one RF cycle, is positive. This axial field depends strongly on both the axial and radial ion coordinates. The superposition of a repulsive potential applied to an exit lens with the diminishing quadrupole potential in the fringing region near the end of a quadrupole rod array can give rise to an approximately conical surface on which the net axial force experienced by an ion, averaged over one RF cycle, is zero. This conical surface has been named the cone of reflection because it divides the regions of ion reflection and ion ejection. Once an ion penetrates this surface, it feels a strong net positive axial force and is accelerated toward the exit lens. As a consequence of the strong dependence of the axial field on radial displacement, trapped thermalized ions can be ejected axially from a linear ion trap in a mass-selective way when their radial amplitude is increased through a resonant response to an auxiliary signal.

High-performance liquid chromatography–tandem mass spectrometry with a new quadrupole/linear ion trap instrument

The use of a new hybrid quadrupole/linear ion trap known as the Q TRAP offers unique benefits as a LC-MS-MS detector for both small and large molecule analyses. The instrument combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. Product ion scans are conducted in a hybrid fashion with the fragmentation step accomplished via acceleration into the collision cell followed by trapping and mass analysis in the Q3 linear ion trap. This results in triple quadrupole fragmentation patterns with no inherent low molecular mass cutoff. In-trap fragmentation is also possible in order to provide triple MS (MS3) capabilities. There are also several scan modes that are not possible on conventional instruments that enable identification of analytes within complex biological matrixes for subsequent high sensitivity product ion scans. This report will describe the new hybrid instrument and the principles of operation, and also provide examples of the unique scan modes and capabilities of the Q TRAP for LC-MS-MS detection in metabolism identification.

On performing simultaneous electron transfer dissociation and collision-induced dissociation on multiply protonated peptides in a linear ion trap

We propose a tandem mass spectrometry method that combines electron-transfer dissociation (ETD) with simultaneous collision-induced dissociation (CID), termed ETD/CID. This technique can provide more complete sequence coverage of peptide ions, especially those at lower charge states. A selected precursor ion is isolated and subjected to ETD. At the same time, a residual precursor ion is subjected to activation via CID. The specific residual precursor ion selected for activation will depend upon the charge state and m/z of the ETD precursor ion. Residual precursor ions, which include unreacted precursor ions and charge-reduced precursor ions (either by electron-transfer or proton transfer), are often abundant remainders in ETD-only reactions. Preliminary results demonstrate that during an ETD/CID experiment, b, y, c, and z-type ions can be produced in a single experiment and displayed in a single mass spectrum. While some peptides, especially doubly protonated ones, do not fragment well by ETD, ETD/CID alleviates this problem by acting in at least one of three ways: (1) the number of ETD fragment ions are enhanced by CID of residual precursor ions, (2) both ETD and CID-derived fragments are produced, or (3) predominantly CID-derived fragments are produced with little or no improvement in ETD-derived fragment ions. Two interesting scenarios are presented that display the flexibility of the ETD/CID method. For example, smaller peptides that show little response to ETD are fragmented preferentially by CID during the ETD/CID experiment. Conversely, larger peptides with higher charge states are fragmented primarily via ETD. Hence, ETD/CID appears to rely upon the fundamental reactivity of the analyte cations to provide the best fragmentation without implementing any additional logic or MS/MS experiments. In addition to the ETD/CID experiments, we describe a novel dual source interface for providing front-end ETD capabilities on a linear ion trap mass spectrometer.

Laser-induced dissociation of singly protonated peptides at 193 and 266 nm within a hybrid linear ion trap mass spectrometer

RATIONALE We implemented, for the first time, laser-induced dissociation (LID) within a modified hybrid linear ion trap mass spectrometer, QTrap, while preserving the original scanning capabilities and routine performance of the instrument. METHODS Precursor ions of interest were mass-selected in the first quadrupole (Q1), trapped in the radiofrequency-only quadrupole (q2), photodissociated under irradiation with a 193- or 266-nm laser beam in the third quadrupole (q3), and mass-analyzed using the linear ion trap. RESULTS LID of singly charged protonated peptides revealed, in addition to conventional amide-bond cleavages, preferential fragmentation at Cα -C/N-Cα bonds of the backbone as well as at the Cα -Cβ /Cβ -Cγ bonds of the side-chains. The LID spectra of [M+H](+) featured product ions that were very similar to the observed radical-induced fragmentations in the CID spectra of analogous odd-electron radical cations generated through dissociative electron-transfer in metal-ligand-peptide complexes or through laser photolysis of iodopeptides. CONCLUSIONS LID of [M+H](+) ions results in fragmentation channels that are comparable with those observed upon the CID of M(•+) ions, with a range of fascinating radical-induced fragmentations.

Off-resonance excitation in a linear ion trap

Off-resonance excitation coupled with mass-selective axial ejection of ions in a linear ion trap is shown to allow coherent control of a trapped ion population. Oscillations of the detected ion current have been found to correspond to the degree of detuning of the excitation field from the resonance frequency. Under appropriate excitation conditions coherent oscillations at the excitation frequency are seen that evolve into the ions’ secular frequency on termination of the excitation field. Termination of the excitation field at various points during the off-resonance excitation profile leaves the ions with different degrees of radial excitation. The degree of radial excitation can be controlled by the coherent excitation field and is demonstrated to be useful for collision-induced dissociation.

Analyzing Glycopeptide Isomers by Combining Differential Mobility Spectrometry with Electron- and Collision-Based Tandem Mass Spectrometry

AbstractDifferential mobility spectrometry (DMS) has been employed to separate isomeric species in several studies. Under the right conditions, factors such as separation voltage, temperature, the presence of chemical modifiers, and residence time can combine to provide unique signal channels for isomeric species. In this study, we examined a set of glycopeptide isomers, MUC5AC-3 and MUC5AC-13, which bear an N-acetyl-galactosamine (GalNAc) group on either threonine-3 or threonine-13. When analyzed as a mixture, the resulting MS and MS/MS spectra yield fragmentation patterns that cannot discern these convolved species. However, when DMS is implemented during the analysis of this mixture, two features emerge in the DMS ionogram representing the two glycopeptide isomers. In addition, by locking in DMS parameters at each feature, we could observe several low intensity CID fragments that contain the GalNAc functionality-specific amino acid residues – identifying the DMS separation of each isomer without standards. Besides conventional CID MS/MS, we also implemented electron-capture dissociation (ECD) after DMS separation, and clearly resolved both isomers with this fragmentation method, as well. The electron energy used in these ECD experiments could be tuned to obtain maximum sequence coverage for these glycopeptides; this was critical as these ions were present as doubly protonated species, which are much more difficult to fragment efficiently via electron-transfer dissociation (ETD). Overall, the combination of DMS with electron- or collision-based MS/MS methods provided enhanced separation and sequence coverage for these glycopeptide isomers. Graphical Abstractᅟ