Applications of collision dynamics in quadrupole mass spectrometry

Some applications of collision dynamics in the field of quadrupole mass spectrometry are presented. Previous data on the collision induced dissociation of ions in triple quadrupole mass spectrometers is reviewed. A new method to calculate the internal energy distribution of activated ions directly from the increase in the cross section for dissociation with center of mass energy is presented. This method, although approximate, demonstrates explicitly the high efficiency of transfer of translational to internal energy of organic ions. It is argued that at eV center of mass energies, collisions between protein ions and neutrals such as Ar are expected to be highly inelastic. The discovery and application of collisional cooling in radio frequency quadrupoles is reviewed. Some previously unpresented data on fragment ion energies in triple quadrupole tandem mass spectrometry are shown that demonstrate directly the loss of kinetic energy of fragment ions in the cooling process. The development of the energy loss method to measure collision cross sections of protein ions in triple quadrupole instruments is reviewed along with a new discussion of the effects of inelastic collisions in these experiments and related ion mobility experiments.     

Collisional focusing effects in radio frequency quadrupoles

The transmission of ions through a conventional two-dimensional radiofrequency-only (rf) quadrupole has been studied for comparatively high operating pressures between 5 × 10^?4 and 1 × 10^?2 torr. Measurements of signals from mass-resolved analyte ions and total ion currents show that, provided the initial injection ion energy is low (1–30 eV), the ion transmission observed through a small aperture at the exit of the rf quadrupole first increases as the gas pressure increases, reaching a maximum at ? 8 × 10^?3 torr before decreasing at higher pressures. This is in direct contrast to the expectations of classical scattering. This “collisional focusing” appears to be analogous to effects seen in three-dimensional ion traps. The collisional focusing increases with the mass of the ion (not mass-to-charge ratio) for masses up to at least 16,950 u. The collisional focusing of the ions is found to be accompanied by significant losses of axial kinetic energy. A Monte Carlo simulation of the energy loss process is reported that can provide agreement with the observed losses for reasonable collision cross-sections. The results suggest that operation of rf quadrupoles at relatively high pressure may find practical application in sampling ions from high (e.g., atmospheric) pressure ion sources.     

Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer

Doubly protonated peptides that undergo an electron transfer reaction without dissociation in a linear ion trap can be subjected to beam-type collisional activation upon transfer from the linear ion trap into an adjacent mass analyzer, as demonstrated here with a hybrid triple quadrupole/linear ion trap system. The activation can be promoted by use of a DC offset difference between the ion trap used for reaction and the ion trap into which the products are injected of 12-16 V, which gives rise to energetic collisions between the transferred ions and the collision/bath gas employed in the linear ion trap used for ion/ion reactions. Such a process can be executed routinely on hybrid linear ion trap/triple quadrupole tandem mass spectrometers and is demonstrated here with several model peptides as well as a few dozen tryptic peptides. Collisional activation of the peptide precursor ions that survive electron transfer frequently provides structural information that is absent from the precursor ions that fragment spontaneously upon electron transfer. The degree to which additional structural information is obtained by collisional activation of the surviving singly charged peptide ions depends upon peptide size. Little or no additional structural information is obtained from small peptides (<8 residues) due to the high electron transfer dissociation (ETD) efficiencies noted for these peptides as well as the extensive sequence information that tends to be forthcoming from ETD of such species. Collisional activation of the surviving electron transfer products provided greatest benefit for peptides of 8-15 residues.     

Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometers in electrospray ionization tandem mass spectrometry of oligonucleotides. 1. Comparison of performance characteristics

The performance of triple-stage quadrupole (TSQ), quadrupole ion trap (QIT), and double focusing sector field (DFSF) mass spectrometers for the generation of fragment ions to obtain sequence information about oligonucleotides was compared. Upon electrospray ionization (ESI), the charge-state distribution of candidate precursor ions not only varied significantly with the type of mass spectrometer, but also with the size and sequence of the investigated oligonucleotides. While concentration limits of detection for an octanucleotide were in the 100 pmol/L range on the QIT and in the 5-10 nmol/L range on the TSQ and DFSF instruments, those of a 24-mer were in the 2-13 nmol/L range on all three instruments. Reproducibility of mass determination, an important prerequisite for reliable identification of fragment ions, was highest on the TSQ with 0.0037% relative standard deviation over three days. Finally, the tandem mass spectra of a dimethoxytritylated pentanucleotide recorded on the three instruments were compared. Relatively simple spectra dominated by complete series of fragment ions of the (a-B) and w type were obtained on the QIT. Complete series of (a-B) and w ions were also observed on the TSQ. However, additional fragments belonging to the b, c, d, x and z series were found in the spectrum. In the spectrum recorded after in-source fragmentation in the DFSF, only fragments corresponding to the loss of a nucleobase and a complete series of w ions were observed. All three mass spectrometers were suitable for the generation of fragment ions, from which the complete nucleotide sequence of the pentanucleotide could be deduced.     

A high pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry ion source for thermal stabilization of labile biomolecules

A high pressure matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) ion source was designed and tested. With this design, pressure is pulsed to an estimated 1-10 mbar in the region of the MALDI sample during desorption with the result of significantly decreased fragmentation compared to similar systems operating with pressures of <0.1 mbar. The thermal stabilization of vibrationally excited ions under these conditions is shown with small peptides desorbed from the "hot" matrix alpha-cyano-4-hydroxycinnamic acid, and with the highly labile oxidized beta-chain of insulin. Fragile gangliosides with several sialic acid residues are desorbed under high pressure and remain intact without the typical losses of sialic acid, and a protein standard, ubiquitin (8565.64 Da), is desorbed with minimal dehydration. Under high pressure collisional cooling conditions, non-covalent matrix adduction to the molecular ions becomes prominent, but with the trapped ions in an FT mass spectrometer, the ions can be mildly activated to detach the matrix adducts. The new source, additionally, generates significant levels of the multiply charged ions which are commonly seen in MALDI-TOFMS, but are rarely observed in MALDI-FTMS. This effect is more likely due to the elimination of a mass filtering effect in the previous FTMS ion source than to collisional cooling of the ions.     

Determination of Cooling Rates in a Quadrupole Ion Trap

Collisional cooling rates of infrared excited ions are measured in a quadrupole ion trap (QIT) mass spectrometer at different combinations of temperature and pressure. Measurements are carried out by monitoring fragmentation efficiency of leucine enkephalin as a function of irradiation time by an infrared laser after a short excitation and incrementally increasing cooling periods. Cooling rates are observed to be directly related to bath gas pressure and inversely related to bath gas temperature. The cooling rate at typical ion trap operating pressure (1 mTorr) and temperature (room T) is faster than can be measured. At elevated temperature and the lowest pressure used for the studies, the rate of collisional cooling becomes negligible compared to the rate of radiative cooling.     

Radial stratification of ions as a function of mass to charge ratio in collisional cooling radio frequency multipoles used as ion guides or ion traps

Collisional cooling in radio frequency (RF) ion guides has been used in mass spectrometry as an intermediate step during the transport of ions from high pressure regions of an ion source into high vacuum regions of a mass analyzer. Such collisional cooling devices are also increasingly used as 'linear', two-dimensional (2D) ion traps for ion storage and accumulation to achieve improved sensitivity and dynamic range. We have used the effective potential approach to study m/z dependent distribution of ions in the devices. Relationships obtained for the ideal 2D multipole demonstrate that after cooling the ion cloud forms concentric cylindrical layers, each of them composed of ions having the same m/z ratio; the higher the m/z, the larger is the radial position occupied by the ions. This behavior results from the fact that the effective RF focusing is stronger for ions of lower m/z, pushing these ions closer to the axis. Radial boundaries of the layers are more distinct for multiply charged ions, compared to singly charged ions having the same m/z and charge density. In the case of sufficiently high ion density and low ion kinetic energy, we show that each m/z layer is separated from its nearest neighbor by a radial gap of low ion density. The radial gaps of low ion population between the layers are formed due to the space charge repulsion. Conditions for establishing the m/z stratified structure include sufficiently high charge density and adequate collisional relaxation. These conditions are likely to occur in collisional RF multipoles operated as ion guides or 2D ion traps for external ion accumulation. When linear ion density increases, the maximum ion cloud radius also increases, and outer layers of high m/z ions approach the multipole rods and may be ejected. This 'overfilling' of the multipole capacity results in a strong discrimination against high m/z ions. A relationship is reported for the maximum linear ion density of a multipole that is not overfilled.     

Controlling the expansion into vacuum—the enabling technology for trapping atmosphere-sampled particulate ions

A new inlet has been designed to control the kinetic energy distributions of ions into a large-radius, frequency-adjusted, linear quadrupole ion trap. The work presented here demonstrates trapping singly-charged, intact proteins in the 10 to 200 kDa range injected from the atmosphere. The trapped ions were held while collisions with a buffer gas removed the remaining amounts of expansion-induced kinetic energy. The ions were then ejected from the trap on-demand into an awaiting detector. There is no low mass limit for ion injection and trapping. The upper limit presented in this study was defined by the limit of the conversion dynode-based detector at ∼1.5 MDa. Trapping larger masses should be achievable. The transmission and capture efficiency across the entire mass range should be very high because the entire flow from the inlet empties directly into the trap. The kinetic energy distribution of massive ions is the primary reason for the working range limitation of mass spectrometers. Trapping ions with collisional cooling before mass analysis permits the motion of the ions to be completely defined by the applied fields. For this reason, this new inlet and trapping system represents a large step toward sensitive, high-resolution mass spectrometry into the megadalton range and beyond.     

Mass spectrometric analysis of leukotriene A4 and other chemically reactive metabolites of arachidonic acid

The biosynthesis of prostaglandins and leukotrienes proceeds through the formation of chemically reactive intermediates leukotriene A4 (LTA4) and prostaglandin H2 (PGH2) which in aqueous solutions have chemical half-lives of 3 s and 3 min, respectively. Prostacyclin (PGI2) is another chemically reactive prostanoid that has a chemical half-life of 3-4 min. The recent development of reversed phase HPLC stationary phases that are stable to elevated pH (pH 10-12) without significant column damage has permitted direct analysis of these acid-sensitive eicosanoids. Using electrospray ionization, molecular anions [M - H]- of these compounds were observed at m/z 317 for LTA4 and m/z 351 for both PGH2 and PGI2. The mechanism of formation of ions derived from collisional activation of LTA4 was studied using stable isotope labeled and chemical analogs of LTA4 and found to involve formation of highly conjugated anions at m/z 261 and 163. The collisional activation of the molecular anion of PGH2 yielded a product ion spectrum identical to that observed for the isomeric prostaglandins PGE2 and PGD2. However, it was possible to baseline separate PGE2, PDG2, and PGH2 by reversed phase HPLC using basic HPLC mobile phases. The collisional activation of PGI2 led to a family of abundant ions including highly conjugated carbon-centered and oxygen-centered radical species (m/z 245 and 205) likely derived from the attack of the carboxylate anion on the cyclic enolether of PGI2 as well as the most abundant product ion (m/z 215) which formed following loss of neutral hexanal and water. The structures of these product ions were consistent with high resolution measurements measured in a quadrupole time-of-flight mass spectrometer.     

Collisional damping interface for an electrospray ionization time-of-flight mass spectrometer

A new interface between atmosphere and high vacuum has been developed for orthogonal injection of electrosprayed ions into a time-of-flight mass spectrometer. A small rf quadrupole operating at 100 mtorr (1.33 × 10^?4 bar) is its key element. Ions enter the quadrupole with velocities acquired in the free expansion/declustering process. As they pass through the quadrupole their motion is constrained by the rf field. Meanwhile, they lose energy by collisions with the gas molecules. The time delays of ions passing through the quadrupole have been measured in order to determine the average velocities of the ions and the factors determining this value. In addition, a simple computational model based on a Monte Carlo approach has been developed to simulate the ion motion; it shows a considerable decrease in both transverse and axial ion velocity components. As the result of collisional damping the interface provides a dramatic improvement in the overall quality of the ion beam transported into the mass spectrometer. Both resolution and sensitivity of the time-of-flight instrument are improved and mass-to-charge ratio discrimination is greatly reduced.     

SORI excitation: Collisional and radiative processes

Theoretical modeling of sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry is described in the present paper. Manipulation of various analytical expressions yield the result that the average laboratory frame collision energy is equal to 2/3 of the maximum kinetic energy in SORI. Survival yields (the fraction of nondecomposed molecular ions) as a function of excitation time, collision energy, and source temperature have been considered: results of MassKinetics-type reaction kinetics modeling were compared with experimental results obtained by Guo et al. (Int. J. Mass Spectrom.2003, 225, 71-82). The results show that radiative cooling has a major influence in SORI-CID. They also suggest that collisional cooling is significant only at very low (less than 0.02 eV) center of mass collision energy; therefore it has a very small influence on the SORI process. Survival yield curves showed excellent agreement between experiments and calculations optimizing two parameters only (collisional energy-transfer efficiency and radiative cooling rate). Using leucine enkephalin as a model compound, the results indicate 0.128 +/- 0.021 energy deposition in a single collision and 7.5 +/- 0.5 s(-1) infrared cooling rate. We also present that these two physical parameters cannot be properly deconvoluted. This effect shows the importance of the parallel consideration of different physical processes.     

飞行时间质谱仪四极杆射频信号放大器的研制

根据自制质谱仪需求研制了一种射频放大装置,由放大电路模块和耦合线圈构成,将其与射频信号发生装置结合用以驱动射频四极杆.该射频放大器可将射频信号放大30倍,为自制射频四极杆提供共振频率1.4 MHz,射频电压峰峰值电压Vp-p可调范围0～600 V的射频信号.自制四极杆用于传输大气压下离子源产生的离子从离子源至工作在高真...     

Pulsed helium introduction into a quadrupole ion trap for reduced collisional quenching during infrared multiphoton dissociation of electrosprayed ions

Previous infrared multiphoton dissociation (IRMPD) experiments utilizing a quadrupole ion trap mass spectrometer yielded limited photodissociation efficiencies. Helium buffer gas continuously infused into the analyzer region at pressures of typically 1 x 10(-3) Torr to improve ion trap performance can collisionally quench photoexcited ions during the IRMPD process. Photodissociation experiments have indicated that uncorrected pressures below 2 x 10(-5) Torr are necessary to avoid collisional deactivation of photoexcited ions. This paper describes IRMPD in the quadrupole ion trap at reduced pressures utilizing a dual-pulsed introduction of helium buffer gas incorporated into the ion trap scan function. The pulsed introduction of helium buffer gas before ion injection allows the efficient trapping of ions injected from an electrospray source and the removal of helium before laser irradiation. A second pulse of helium directly before ion detection improves the intensity of the ion signal. The use of this dual-pulsed inlet of helium for improved IRMPD is demonstrated with the carbohydrate antibiotics neomycin and erythromycin. Copyright 2000 John Wiley & Sons, Ltd.     

Study of H/D-exchange reaction kinetics of polypeptides

One of the possible methods for 3D protein structure investigation is the study of gas-phase hydrogen/deuterium (H/D) exchange reaction between protein ions and a D-containing reactant gas, e.g., D₂O or ND₃. A segmented radio frequency quadrupole (RFQ) was used as a molecule-ion reactor to study gasphase H/D-exchange of protonated ions of three different peptides. The ions were produced in an electrospray ion source. The RFQ is a part of ion transport interface of a high resolution orthogonal time-of-fiight mass spectrometer (O-TOF MS). The RFQ was modified for a linear ion trap (LIT) mode of operation to increase a dwell time of target ions inside the RFQ. Phase-sensitive operation of the LIT and the O-TOF MS was controlled by custom developed PC executive program. The reaction mixture of N₂ and ND₃ was injected into the reactor, while keeping its partial pressure in the range of 10⁻³–10⁻² mbar, and corresponding ND3 concentration in the range of 10¹³–10¹⁴ cm⁻³. It was possible to vary the ion dwell time in the reactor between 30 ms and 1 s. H/D-exchange was studied for leucine enkephalin, gramicidin S and apamin. The data analysis based on statistical approach has shown a principal possibility to distinguish different mobile H-atoms of peptides, taking part in H/D-exchange, according to reaction rates.     

Separation and sweeping of metal ions with EDTA in CZE-ESI-MS

Ethylenediaminetetraacetic acid (EDTA) in the background electrolyte (BGE) of capillary zone electrophoresis coupled to an electrospray ionization mass spectrometer is presented as an approach for the determination of metal ions. Significant signals for the metal-ligand complexes were observed even when EDTA was continually eluted from the capillary during the entire electrophoretic run. The signal-to-noise ratio was improved by the addition of ammonia to the sheath liquid and by using an acquisition m/z range above the m/z of EDTA. The LODs for the test metal ions (i.e. calcium(II), manganese(II), and zinc(II)) with conventional injection were around 1-2 mg/L with corrected peak areas that are linear from 8 to 100 mg/L. The presence of EDTA in the BGE was critical not only for the separation but also for sweeping via complexation as an on-line sample concentration technique. The peak height of the test metal ions was improved at least tenfold with sweeping via EDTA complexation and yielded LODs in the μg/L range.     

Practical considerations when using radio frequency-only quadrupole ion guide for atmospheric pressure ionization sources with time-of-flight mass spectrometry

Construction details and performance evaluation of a radio frequency (rf)-only quadrupole ion guide for use with an electrospray ionization time-of-flight mass spectrometer is presented in this paper. Angiotensin III and cytochrome c were used in these experiments to investigate the ion transmission properties of the rf-only quadrupole for different m/z species. In addition, influence of ion kinetic energies along with the characteristic fragmentation due to collision induced dissociation (CID) were studied. These experiments demonstrate that the transmissions of different m/z ions were not only dependent on the frequency and magnitude of the rf waveform, which is similar to a high vacuum rf-only quadrupole ion guide, but also on the pressure inside the quadrupole chamber. For the pressure range tested, low m/z ions are better focused with increasing pressure. As expected, transmission of ions are subject to space charge limitations when significant numbers of ions are focused on the axis of the quadrupole. It is also observed that CID results are related to transverse motion and longitude motion of ions inside the quadrupole region. Consequently, CID is useful for fragmentation of linear peptides and it is not effective (in present configuration) for large bulky proteins. The kinetic energy of ions that enter the repelling region of the TOFMS is ultimately determined by the ensemble effect resulting from the dc bias potential of the quadrupole (the dominant factor), skimmer-2, pressure inside the quadrupole chamber, and jet expansion. While this system is tested with an ESI source, the operational principle and design criteria are directly applicable for improving other atmospheric pressure ionization sources with time-of-flight mass analyzers such as an inductively coupled plasma ion source.     

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.     

Simulation of the Collisional Cooling Effect in a Quadrupole Ion Trap Mass Spectrometer

Examination of the collisional cooling effect of the buffer gases on ion trapping and detection in an ion trap mass spectrometer has been undertaken by the SIMION 3D program. Computation for the kinetic energy of ions under various conditions was used to account for the effects of collisional cooling of ions. Several parameters that may affect the collisional cooling effects of ions are evaluated including the existence and the variation of pressure of the buffer gas; the temperature of the ion trap; the size of the inner radius of the ion trap electrodes; the mass to charge ratio of ions; the alternative buffer gases and the q_z. values which establish the ion trap trapping environment.     

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.