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This work presents the experimental evaluation of a digital tandem mass filter that is composed of two digitally operated low-resolution mass filters in series whose mass windows are shifted with respect to each other. The overlap of the mass windows allows the resolution (Δ m ) of ions to be narrowed to provide better resolving power, while the acceptance of the tandem mass filter is defined by the acceptance of the first low-resolution quadrupole. Our experiments show that digital operation fulfills the promise of the tandem mass filter for providing better ion transmission at the same or better resolving power as a single quadrupole mass filter. It allows the user to continuously adjust the resolving power and sensitivity to meet current needs. Most importantly, the observed resolving power/sensitivity characteristics are the same at any mass and m / z .

Rationale: In the development of commercial quadrupole mass spectrometers, there is an interest in understanding the factors that affect the transmission and peak shape of ions passing through the mass filter. These include the ion collection effects of the ion optics that lead to the presence of a peak structure., Methods: The peak structure can be observed by increasing the ion's axial kinetic energy and reducing the mass step size in a mass spectrum. The calculation of the maxima in the peak structure can be achieved by first obtaining the Mathieu characteristic parameter β y from the scan lines for each mass. The ion's secular frequency, ω 0,y , along with the ion's transit time through the mass filter can then be used to calculate the location of the maxima in the peak structure., Results: Experimentally, a peak structure has been observed for m/z 42, m/z 118 and m/z 622 in the Q1 scan mode of a triple quadrupole mass spectrometer. The peak structure has shown a dependency on the ion's axial kinetic energy, but, a mass independency with regard to the number of maxima is observed in the peak structure. In the Q3 scan mode, there is an absence of the peak structure due to the ion collection efficiency of the HED detection system., Conclusions: Under the right conditions, the peak structure due to ion collection effects can be observed with a quadrupole mass filter operated in the Mathieu first stability region. The presence of the peak structure has been shown to depend upon the ion collection effects at the exit of the mass filter., (© 2022 John Wiley & Sons Ltd.)

Rationale: Ion reflections at the quadrupole pre-filter/mass filter boundary lead to the trapping of ions within the pre-filter. The trapped ions lead to space charge effects on the transmission of ions through this region which become more of an issue with the development of high sensitivity mass spectrometers which have bright ions beams., Methods: Simulations were carried out matching the effective potentials at the pre-filter/mass filter boundary to reduce the field gradients across the boundary by reducing the field radius of the pre-filter. A further rotation of the pre-filter relative to the mass filter was incorporated into the model. Experiments were carried out to reproduce the conditions of the simulations., Results: Simulations show that matching the effective potentials at the pre-filter/mass filter boundary and rotating the pre-filter by 45° reduced the number of reflected ion trajectories when the mass filter was operated in either the radiofrequency (RF)-only mode or in mass resolving mode. Experiments confirmed the reduced effects of space charge on the transmission of ions through the pre-filter through the measurement of pre-filter transmission profiles with and without the use of an empty step to remove any trapped ions. These results showed significant improvement over the typical pre-filter/mass filter set-up., Conclusions: Matching the effective potentials at the quadrupole pre-filter/mass filter boundary and rotating the pre-filter result in a pre-filter/mass filter set-up that does not lead to ions that are reflected at the boundary. The modified set-up does not result in the presence of unwanted trapped ions in the quadrupole pre-filter., (© 2019 John Wiley & Sons, Ltd.)

Rationale: Quadrupole pre-filter transmission profiles are shown to be affected by the presence of trapped ions within the pre-filter of a quadrupole mass spectrometer. Trapped ions are the result of ions reflected at the pre-filter/mass filter boundary and lead to space charge effects that can affect the shape of the pre-filter transmission profile, ion beam stability, peak width and peak intensity. The development of high sensitivity mass spectrometers with bright ion beams leads to greater numbers of trapped ions., Methods: Experimental pre-filter transmission profiles have been measured with and without an empty step to clear the pre-filter region of trapped ions showing the extent that space charge has on ion transmission through the pre-filter. Ion trajectory simulations, using a restricted set of initial conditions, have been used to help understand the structure of the pre-filter transmission profile., Results: Experiments show that the effects of trapped ions within the pre-filter can be easily seen in the shape of the pre-filter transmission profile, ion beam stability and peak shape. The pre-filter transmission profile shows structure which is related to the secular frequency of the ion of interest which is related to the Mathieu parameter q of the pre-filter. Simulations show that the nodes observed in the experimental transmission profile are spaced by half the secular period of the ion., Conclusions: The structure of the pre-filter transmission profile can be observed experimentally when steps are taken to remove trapped ions from within the pre-filter. A better understanding of the factors contributing to the shape of the pre-filter transmission profile from simulations has been demonstrated., (© 2019 John Wiley & Sons, Ltd.)

Rationale: A high dynamic range pulse counting system has been developed that demonstrates an ability to operate at up to 2e8 counts per second (cps) on a triple quadrupole mass spectrometer. Previous pulse counting detection systems have typically been limited to about 1e7 cps at the upper end of the systems dynamic range. Modifications to the detection electronics and dead time correction algorithm are described in this paper., Methods: A high gain transimpedance amplifier is employed that allows a multi-channel electron multiplier to be operated at a significantly lower bias potential than in previous pulse counting systems. The system utilises a high-energy conversion dynode, a multi-channel electron multiplier, a high gain transimpedance amplifier, non-paralysing detection electronics and a modified dead time correction algorithm. Modification of the dead time correction algorithm is necessary due to a characteristic of the pulse counting electronics., Results: A pulse counting detection system with the capability to count at ion arrival rates of up to 2e8 cps is described. This is shown to provide a linear dynamic range of nearly five orders of magnitude for a sample of aprazolam with concentrations ranging from 0.0006970 ng/mL to 3333 ng/mL while monitoring the m/z 309.1 → m/z 205.2 transition. This represents an upward extension of the detector's linear dynamic range of about two orders of magnitude., Conclusions: A new high dynamic range pulse counting system has been developed demonstrating the ability to operate at up to 2e8 cps on a triple quadrupole mass spectrometer. This provides an upward extension of the detector's linear dynamic range by about two orders of magnitude over previous pulse counting systems., (Copyright © 2013 John Wiley & Sons, Ltd.)

An exploration of the parameters necessary to obtain high-resolution excitation, using dipolar excitation, of an ion in a linear ion trap has been undertaken in this study. These parameters included ion trap pressure, excitation amplitude, excitation period, drive frequency of the ion trap, Mathieu q value and the mass of the ion of interest. An understanding of how these parameters play a role in high-resolution excitation is necessary to the development of a method for the targeted tandem mass spectrometric (MS/MS) analysis of ions with the same nominal mass. Resonance excitation profiles with full width half maxima as narrow as 0.015 m/z units could be obtained, under the right conditions, for an ion from a homogenously substituted triazatriphosphorine at m/z 322.049, which translates into a mass resolution of >21 500. In this particular case the requirement for high resolution was a low trap pressure (3.8 × 10(-5) Torr), low excitation amplitude (3 mV), long excitation period (100 ms) and a high Mathieu q value(0.8) when using a drive frequency of 1.228 MHz. Similar conditions were used to demonstrate the isolation of individual [M + H](+) component ions from mixtures of bromazepam (m/z 316.008)/chlorprothixene (m/z 316.0921)/fendiline (m/z 316.206) and chlorprothixene (m/z 316.0921)/oxycodone (m/z 316.1543)/fendiline (m/z 316.206) prior to obtaining product ion spectra with excitation at q = 0.236. In the former mixture the individual components were isolated with near 100% efficiency while in the latter mixture the isolation efficiency dropped to near 50% for the oxycodone component and to 80% for the other components., (Copyright © 2010 John Wiley & Sons, Ltd.)

A pulsed valve was used to increase the pressure within the trapping region of a low-pressure linear ion trap by situating the pulsed valve close to the ion trapping region. The pressure was estimated to increase from a background pressure of 3.5e-5 Torr of nitrogen to 0.49 mTorr at the center of the trap. The increased pressure allowed excitation periods to be reduced from 100 to 25 ms without suffering losses in MS/MS efficiency during dipolar excitation. The reduction in excitation period translates into an increase in the overall duty cycle of the scan.

The efficiency of in-trap fragmentation in a low-pressure linear ion trap (LIT), using dipolar excitation, is dependent upon the choice of both the excitation q and the drive frequency of the quadrupole. In the work presented here, fragmentation efficiencies have been measured as a function of excitation q for drive frequencies of 816 kHz and 1.228 MHz. The experiments were carried out by fragmenting reserpine (609.23-->448.20 Th and 397.21-->365.19 Th transitions) and caffeine (195-->138 Th and 138-->110 Th transitions). The data showed that the onset of efficient fragmentation occurred at a lower Mathieu q for the LIT operated at 1.228 MHz when compared with the LIT operated at 816 kHz. A comparison of the fragmentation efficiency curves as a function of pseudo-potential well depth showed that the onset of fragmentation is independent of the drive frequency. In addition, a comparison of the fragmentation efficiency curves showed that all four of the precursor ions fragmented within a range of four V of pseudo-potential well depth. The choice of an appropriate excitation q can then be determined based upon a minimum pseudo-potential well depth, quadrupole field radius, drive frequency, and the mass of interest. Fragmentation efficiencies were also found to be significantly greater when using the higher drive frequency.

It has been shown that through the process of resonant excitation the fragmentation of ions confined in a low-pressure (<0.05 mTorr) linear ion trap (LIT) can be accomplished while maintaining both high fragmentation efficiency and high resolution of excitation. The ion reserpine, 609.23 Da, has been fragmented with efficiencies greater than 90% while a higher mass ion, a homogeneously substituted triazatriphosphorine of mass 2721.89 Da, has been fragmented with 48% efficiency. This was accomplished by extended resonant excitation by low-amplitude auxiliary RF signals. Computer modelling of ion trajectories and analysis of the trapping potentials have demonstrated that a reduction in neutralization of ions on the rods (or losses on the rods) and increased fragmentation is a consequence of higher order terms in the potential introduced by the round-rod geometry of the LIT.

Experiments examining the excitation of the quadrupolar n = 0, K = 1 to 6 resonances for the ion reserpine in a linear ion trap have been shown to produce resonance shifts that were dependent upon either or both of the excitation amplitude and trap pressure (Collings, B. A.; Douglas, D. J. J. Am,. Soc. Mass Spectrom., 2000, 11, 1016-1022). The extent of this dependency was determined by examining the effects of each parameter using an ion trajectory simulator. The simulations indicated that it is the change in excitation amplitude that is mostly responsible for the resonance shifts with a minor dependency upon the trapping pressure. It was found that the higher excitation amplitudes required to observe the higher order resonances resulted in greater shifts relative to the theoretical resonances predicted for an excitation amplitude of zero volts. The nature of these shifts can be understood by examining the equations of motion for an ion trapped in a quadrupolar potential during the excitation process. Rearrangement of the equations of motion lead to a Mathieu stability diagram in which the coordinate and ordinate variables are dependent upon the excitation frequency and amplitude. In such a diagram the resonances occur in the regions of instability. The calculated resonance shifts showed good correlation with the experimental and simulation results.

A detailed description of a linear ion trap time-of-flight (TOF) mass spectrometer system, capable of sequential mass spectrometry (MS(n)), is given. Many improvements have been incorporated since the initial description of this system (Rapid Commun. Mass Spectrom. 1998; 12: 1463-1474). The pressure in the trap has been lowered from 7.0 to 1.8 mTorr, resulting in an increase in the mass resolution of ion excitation from 75 to 240. Use of the system for MS(3) is demonstrated. Dipole excitation of the n = 1 harmonic, instead of the n = 0 fundamental frequency of ion motion, is shown to have a higher frequency resolution, f/Deltaf, but lower mass resolution, m/Deltam. Both experiments and modeling demonstrate that at the lower pressure there is less collisional cooling of ions in the axial and radial directions of the trap. The efficiency of trapping is shown to be nearly 100% for periods up to 5 s. The demonstrated mass range for mass analysis has been extended to greater than m/z 16 250. To avoid the formation of adduct ions when trapping protein ions for extended times requires ultra-high vacuum cleanliness conditions, even though the trap operates in the mTorr-pressure range. Upgrading the TOF to a reflectron with higher quality ion optics results in an increase in the mass resolution of the TOF mass spectrometer to about 5000 at m/z 750., (Copyright 2001 John Wiley & Sons, Ltd.)

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for detection of noncovalent complexes. However, detection of noncovalent protein-protein complexes is not a widespread practice and still produces some challenges for mass spectrometrists. Here we demonstrate the detection of a noncovalent protein-protein complex between alpha-amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha-amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic alpha-amylase and its microbial inhibitor tendamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over enzyme strongly affect the detection of noncovalent complexes in the gas phase. ESI mass spectra of alpha-amylase at pH 7 exhibited charge states significantly lower than that reported previously, which is indicative of a native protein conformation necessary to produce a noncovalent complex. Detection of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some cases even quantitative, characterization of equilibria of noncovalent complexes in solution is possible., (Copyright 2001 John Wiley & Sons, Ltd.)

The resonant frequencies for quadrupole excitation of ions confined with a buffer gas in a linear quadrupole ion trap with Mathieu parameters a = 0 and q approximately 0.36 have been measured. The resonances are predicted to occur at angular frequencies omega(K)n given by omega(K)n = (omega/K)n + beta without the presence of a buffer gas where omega is the angular frequency of the trapping radio frequency, K = 1, 2, 3... is the order of the resonance calculated with perturbation theory, and n = 0, +/-1, +/-2, +/-3.... The resonances are measured through fragmentation of protonated reserpine. The observed frequencies agree closely with the theoretical values but there are small differences which vary from +0.6% at K = 2 to -2.7% at K = 6. This is believed to be the result of the dependence of the resonant frequencies upon the buffer gas density and/or the excitation amplitude. The resolution of the resonances (measured from the depletion of precursor and formation of fragment ions) increased by a factor of 2 as K increased from 1 to 6. This increase in resolution warrants further investigation into the use of higher order resonances for isolation and excitation of trapped ions.

The properties of a quadrupole mass filter operated in the fourth stability region with Mathieu parameters (a,q) = (2 x 10(-3), 21.3) have been investigated experimentally and theoretically. A resolution at half-height (R1/2) of 13,900 has been obtained for 40 eV 39K+ ions formed by thermal ionization. With ion energies of 750 eV or more, a resolution at half-height of up to 5000 can be obtained. Extrapolation of resolution data obtained with ions of 40 eV-4800 eV energy shows that unit resolution should be possible for ions with energies of approximately 10 keV. For low-energy ions (e.g., 75 eV), the transmission of the quadrupole operated in the fourth region is many orders of magnitude less than that of a quadrupole operated in the first or second stability regions. Acceptance calculations show this is a result of the combination of a much lower acceptance and strongly defocusing fringing fields. However, for higher ion energies, the transmission with operation in the fourth region is found to be comparable to that of a quadrupole operated at the same resolution in the second region. The implications for inductively coupled plasma mass spectrometry are briefly discussed.

Collision cross sections have been measured for gas phase ions of native oxidized bovine pancreatic trypsin inhibitor (BPTI), native reduced BPTI and a mutant form of BPTI containing a single disulphide bond between residues 5 and 55 ([5-55]Ala BPTI). Cross sections for [5-55]Ala BPTI and reduced BPTI were 9% and 17% greater respectively than those for native BPTI. Cross sections for native BPTI were smaller than previous estimates from the crystal structure but in reasonable agreement with values calculated from the radius of gyration determined by x-ray scattering from solution BPTI. The increase in cross section for reduced BPTI over native BPTI is similar to that seen in molecular dynamics simulations. The results show that the disulphide bonds of BPTI contribute to the folding of the gas phase ions, but that even in the absence of disulphide bonds the protein ions maintain compact structures. Comparisons to relative areas calculated from hydrodynamic radii of BPTI in solution suggest that when disulphide linkages are removed, BPTI in the gas phase unfolds less than BPTI in solution.

Gas phase holomyoglobin (hMb) ions in charge states +7 to +21 were formed by electrospray ionization in combination with a continuous-flow mixing apparatus. Collision cross section measurements show that the highly charged ions are somewhat unfolded in comparison to low charge states but still retain a considerable degree of folding. A new collision model is presented which calculates the relative energies transferred to complexes in tandem mass spectrometry. Tandem mass spectrometry and ion trapping experiments both show that the energies required to dissociate heme from the highly charged heme-protein complexes in the gas phase are similar to those of low charge states, previously shown in literature ion cyclotron resonance experiments to be 0.7-1.0 eV. These energies are comparable to those of the heme binding energy in solution. The results suggest that even for the highly charged hMb ions which have unfolded somewhat, the heme-protein interactions remain relatively unperturbed.

A new method for studying the folding kinetics of proteins is described. The method combines a continuous flow mixing technique with an electrospray mass spectrometer. Different protein conformations in solution are detected by the different charge states they produce during electrospray ionization. Unfolded proteins generally have more accessible protonation sites and give higher charge states than native proteins. The method is applied to study the refolding of acid-denatured cytochrome c. Global data analysis is used to obtain the exponential lifetimes which are associated with the refolding process. The kinetics can be described by two lifetimes of 0.17 +/- 0.02 and 8.1 +/- 0.9 s which are in accordance with the results of stopped flow experiments previously described in the literature. These lifetimes are associated with roughly 90 and 10% of the total intensity changes in the mass spectrum, respectively, and most likely reflect fast and slow refolding subpopulations of cytochrome c in solution.