Fourier transform ion cyclotron resonance mass spectrometer with coaxial multi-electrode cell ('O-trap'): first experimental demonstration.

The conceptual design of the O-trap Fourier transform ion cyclotron resonance (FT-ICR) cell addresses the speed of analysis issue in FT-ICR mass spectrometry. The concept of the O-trap includes separating the functions of ion excitation and detection between two different FT-ICR cell compartments. The detection compartment of the O-trap implements additional internal coaxial electrodes around which ions with excited cyclotron motion revolve. The expected benefits are higher resolving power and the lesser effect of the space charge. In this work we present the first experimental demonstration of the O-trap cell and its features, including the high ion transfer efficiency between two distinct compartments of an ICR cell after excitation of the coherent cyclotron motion. We demonstrate that utilization of the multiple-electrode detection in the O-trap provides mass resolving power enhancement (achieved over a certain time) equal to the order of the frequency multiplication. In an O-trap installed in a 5 T desk-top cryogen-free superconducting magnet, the resolving power of R = 80,000 was achieved for bradykinin [M + 2H](2+) (m/z 531; equivalent to 100,000 when recalculated for m/z 400) in 0.2 s analysis time (transient length), and R = 300,000 at m/z 531 for a 1 s transient. In both cases, detection on the third multiple of the cyclotron frequency was implemented. In terms of the acquisition speed at fixed resolving power, such performance is equivalent to conventional FT-ICR detection using a 15 T magnet.

Ideal velocity focusing in a reflectron time-of-flight mass spectrometer.

A single-stage ion mirror in a time-of-flight (TOF) mass spectrometer (MS) can perform first order velocity focusing of ions initially located at a start focal plane while second order velocity focusing can be achieved using a double-stage reflectron. The situation is quite different when an ion source extraction field is taken into account. In this case which is common in any practical matrix-assisted laser desorption/ionization (MALDI) TOF-MS a single-stage reflectron, for example, cannot perform velocity focusing at all. In this paper an exact, analytic solution for an electric field inside a one-dimensional reflectron has been found to achieve universal temporal focusing of ions having an initial velocity distribution. The general solution is valid for arbitrary electric field distributions in the upstream (from the ion source to the reflectron) and downstream (from the reflectron to an ion detector) regions and in a decelerating part of the reflectron of a reflectron TOF mass spectrometer. The results obtained are especially useful for designing MALDI reflectron TOF mass spectrometers in which the initial velocity distribution of MALDI ions is the major limiting factor for achieving high mass resolution. Using analytical expressions obtained for an arbitrary case, convenient working formulas are derived for the case of a reflectron TOF-MS with a dual-stage extraction ion source. The special case of a MALDI reflectron TOF-MS with an ion source having a low acceleration voltage (or large extraction region) is considered. The formulas derived correct the effect of the acceleration regions in a MALDI ion source and after the reflectron before detecting ions.

A quadrupole ion trap/time-of-flight mass spectrometer with a parabolic reflectron.

A matrix-assisted laser desorption/ionization-quadrupole ion trap/reflectron time-of-flight (MALDI-QIT/reTOF) mass spectrometer design and its operation in both normal and tandem mass spectrometric modes are described. A parabolic reflectron was found to be capable of providing mass resolution of 5000 for an initial ion energy distribution ranging over a 50% energy interval of the entire reflectron energy range. The sensitivity, ion isolation and fragmentation efficiency in the MALDI-QIT/reTOF instrument were close to those observed in the MALDI/QIT mass spectrometer. The mass resolution was shown to depend on the extraction field potentials, the r.f. trapping voltage amplitude and the phase of shutting down the r.f. voltage before extraction. At values of qs < 0.3-0.4 the mass resolution does not depend on the ion mass, is in a range of 1000-1400 and is governed by the extraction voltages and the ion temperature before extraction, the latter shown to be in the range 1180-1690 K. The variation of the mass resolution for ions at values of qs > 0.4 is irregular but normally it is lower than that for ions having lower qs values. Mass spectral line positions shifted when the trapping voltage before extraction was varied. The line shifts were larger for lower mass ions and were comparable to the line widths in the case of very low masses.

Injection of externally generated ions into an increasing trapping field of a quadrupole ion trap mass spectrometer.

Trapping ions injected into a quadrupole ion trap (QIT) by increasing the trapping r.f. voltage on a ring electrode is an effective and widely recognized method of interfacing an ion trap with pulsed ion sources such as matrix-assisted laser desorption/ionization (MALDI). In this paper, the problem of mass discrimination during the injection and trapping of ions by the increasing r.f. field was studied both experimentally and by numerical simulation using SIMION software. For a MALDI/QIT interface design with a remote external ion source described here, experiments with polyethylene glycol (PEG 1000 and PEG 1500) showed little mass discrimination for trapping ions in a wide mass range (500-2000 Dn) for a broad range of experimental conditions, which include kinetic energies of 5-40 eV for the injected ions and an r.f. voltage of 400-4000 Vo-p amplitude ramped at a rate of 30-140 Vo-p mus-1. In the numerical simulation, complex and sharp dependences of the trapping efficiency on the phase of the r.f. voltage and initial kinetic energy of ions were observed. However, after averaging over the r.f. phase and over a reasonable range of kinetic energy, the simulation resulted in relatively constant and high values for the trapping efficiency (normally 0.2-0.3) for any mass and kinetic energy considered, which are consistent with the weak sensitivity to injection parameters observed in the experiment. A simple model for the qualitative description of ion injection and trapping is suggested that relies on phase interaction of injected ions with the r.f. field rather than on collisions with the buffer gas molecules to decrease the ion kinetic energy.

Gas phase hydrogen/deuterium exchange reactions of peptide ions in a quadrupole ion trap mass spectrometer.

Hydrogen/deuterium exchange reactions of protonated and sodium cationized peptide molecules have been studied in the gas phase with a MALDI/quadrupole ion trap mass spectrometer. Unit-mass selected precursor ions were allowed to react with deuterated ammonia introduced into the trap cell by a pulsed valve. The reactant gas pressure, reaction time, and degree of the internal excitation of reactant ions were varied to explore the kinetics of the gas phase isotope exchange. Protonated peptide molecules exhibited a high degree of reactivity, some showing complete exchange of all labile hydrogen atoms. On the contrary, peptide molecules cationized with sodium exhibited only very limited reactivity, indicating a vast difference between the gas phase structures of the two ions.

Pulsed gas introduction for increasing peptide CID efficiency n a MALDI/quadrupole ion trap mass spectrometer.

A pulsed valve was used for studying the effects of introducing heavy gases at different stages of operation of a quadrupole ion trap and for increasing the efficiency of collision-induced dissociation (CID) of peptide ions at low values of the Mathieu parametere qz. When amounts of heavy gases comparable to that of the helium buffer gas were introduced during the ion trapping, ion isolation, and mass spectral recording stages, the effects on performance were generally small or negative. However, injection of heavy gases during CID provided considerable improvement in fragmentation efficiency that depended upon the particular gas used, its mass and pressure, and the amplitude of the excitation voltage. Efficient peptide fragmentation could be demonstrated for values of qz as low as 0.05, which permitted trapping of low-mass product ions and (in many cases) full recovery of the amino acid sequence. In this report, examples are provided of monoisotopic tandem mass spectra of peptide ions with masses up to 1570 Da.

Advanced stored waveform inverse Fourier transform technique for a matrix-assisted laser desorption/ionization quadrupole ion trap mass spectrometer.

The stored waveform inverse Fourier transform (SWIFT) technique is used for broadband excitation of ions in an ion-trap mass spectrometer to perform mass-selective accumulation, isolation, and fragmentation of peptide ions formed by matrix-assisted laser desorption/ionization. Unit mass resolution is achieved for isolation of ions in the range of m/z up to 1300 using a two-step isolation technique with stretched-in-time narrow band SWIFT pulses at the second stage. The effect of 'stretched-in-time' waveforms is similar to that observed previously for mass-scan-rate reduction. The asymmetry phenomenon resulting from the stretched ion-trap electrode geometry is observed during application of normal and time-reversed waveforms and is similar to the asymmetry effects observed for forward and reverse mass scans in the resonance ejection mode. Mass-selective accumulation of ions from multiple laser shots was accomplished using a method described earlier that involves increasing the trapping voltage during ion introduction for more efficient trapping of ions.

Effect of phase locking AC and RF voltages for high mass analysis in a quadrupole ion trap mass spectrometer.

Experiments on the influence of locking the AC and RF frequency phases on the mass resolution and line position in the mass spectra from a quadrupole ion trap mass spectrometer were carried out at low ejection values of beta z to investigate the possibility of using this effect for high mass analysis. At a typical mass scan rate of 1000 u/s the improvement for the mass resolution in these experiments did not exceed 10-20% at beta z = 0.25 compared with 200-250% at beta z = 0.5. However, without phase locking, the mass resolution at beta z = 0.25 was about two times higher than that at beta z = 0.5. Thus, the absolute values for the mass resolution observed without phase-locked AC and RF frequencies at beta z = 0.25 were about the same as at beta z = 0.5 with phase locking. This observation was explained by a significant negative contribution to the mass resolution of the ion microoscillations along the trajectory at the fundamental RF frequency which is different for the cases of beta z = 0.25 and beta z = 0.5.

High-performance collision-induced dissociation of peptide ions formed by matrix-assisted laser desorption/ionization in a quadrupole ion trap mass spectrometer.

A modified ion trap detector has been utilized to obtain high-performance collision-induced dissociation (CID) mass spectra of peptide ions formed by matrix-assisted laser desorption/ionization (MALDI). MALDI ions are trapped while increasing the fundamental radio frequency field, obviating the need for elevated helium gas pressures. Molecular ion isotopic clusters are then isolated by a reverse-forward-reverse scan sequence. A single species within the isotopic cluster (generally the monoisotopic mass) is then selected for activation. Finally, modulation of the amplitude of the resonant excitation voltage on the end-cap electrodes, used previously to improve mass calibration in normal mass spectra, is now utilized to provide high mass accuracy for the product ions. The CID mass spectra of several protonated and sodium-cationized peptides are presented and are often characterized by a series of rearrangement ions that can be utilized in the determination of amino acid sequences.

Sequencing electroblotted proteins by tandem mass spectrometry.

Electroblotting proteins separated by gel electrophoresis provides a suitable support for further manipulations and analysis of small amounts of relatively pure samples. On-membrane digestion, peptide mapping by mass spectrometry, and database searching offer sensitive and fast tools to identify the analyte. By providing sequence information, tandem mass spectrometry can go a step further, confirming the database identification, solving problems connected with post-translational modifications and sequence variations, or supplying the stretches of internal sequence necessary to synthesize an oligonucleotide probe for gene isolation. The viability of this approach was successfully evaluated using different tandem mass spectrometric techniques: metastable decomposition in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight instrument with a curved-field reflectron; low energy collision-induced dissociation in a MALDI quadrupole ion trap mass spectrometer; and high energy collision-induced dissociation in a high-performance four-sector mass spectrometer with massive cluster-impact ionization.