Spontaneous and deflected drift-trajectories in orthogonal acceleration time-of-flight mass spectrometry

Orthogonal acceleration is a method for gating ions from an ion beam into a time-of-flight (TOF) mass spectrometer. The technique involves a pulsed electric field to apply acceleration directed orthogonally to an ion beam. This approach is useful for coupling continuous ion sources to TOF mass analyzers. Most instruments of this type, which have been described in the literature, use steering electrodes after the orthogonal acceleration step. Those velocity components of ions originating from the ion beam velocity are minimized so that the deflected drift-trajectory is parallel to a transverse flight tube. In an alternative geometry the ion beam velocity is conserved and the drift-trajectory after the orthogonal acceleration step is spontaneous. The differences between the space-time focusing ability with spontaneous and deflected drift-trajectories are discussed and investigated. Trajectory calculations indicate that deflection fields placed after the orthogonal acceleration step distort the ion packet because, in this geometry, the flight-time to the detector is dependent on the position that the ions enter the steering optics. Increasing the duty-cycle efficiency by sampling longer sections of the continuous ion beam leads to a degradation of resolving power. Employing a spontaneous drift-trajectory after orthogonal acceleration provides the advantage that the arrival time spread for isobaric ions is, in principle, independent of the length of the ion beam sampled. The major implication of these findings is that simultaneously optimized sensitivity and resolving power may not be achievable with the deflected drift-trajectory instruments. The calculations are in agreement with results from the published data of a number of groups who have built instruments based on the orthogonal acceleration principle.     

Investigation of the advanced functionalities of a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer

Time-of-flight mass spectrometry (ToF-MS) has gained wide acceptance in many fields of chemistry, proteomics, metabolomics and small molecule analysis. ToF-MS, however, has some inherent advantages and drawbacks. Numerous developments have been made to hybrid ToF instruments to improve their capabilities. We have used a quadrupole orthogonal acceleration ToF (Q-oa-ToF) instrument to assess developments made to improve resolution, dynamic range and signal-to-noise (S/N) ratios (i.e. sensitivity). Higher mass resolution can improve the analysis of mixtures containing compounds with similar m/z values and improved mass accuracy gives greater confidence for structural elucidation applications. Wide dynamic ranges are necessary for the analysis of unknown samples or samples that vary widely in analyte concentrations. The performance of the advanced functionalities for routine structural elucidation in terms of resolution, dynamic range and S/N ratios was investigated using test compounds. The results presented in this work demonstrate and validate the use of these new enhancements for Q-ToF instruments and also show their limitations.     

Multi-element analysis of urine by inductively coupled plasma orthogonal acceleration time-of-flight mass spectrometry

This work shows the analytical potential of inductively coupled plasma orthogonal-acceleration time-of-flight mass spectrometry (ICP-OA-TOF-MS) for rapid, simultaneous, and reliable determination of more than 50 elements at ultra-trace levels in urine. Under optimum instrumental conditions, after a 10-fold sample dilution step, and by using Rh as an internal standard, ICP-OA-TOF-MS also enables the determination of elements whose assay is more diffcult when using conventional quadrupole instruments. This is confirmed by the analysis of commercially available reference urine samples and/or by analytical recoveries study and isotope ratio based determination of accuracies. On the other side, the interference resulting from polyatomic carbon, chlorine, or various sulfur species does not allow the determination of elements such as Cr, Fe, V, Se and As without a mathematical correction.

Improvement of the duty cycle of an orthogonal acceleration time-of-flight mass spectrometer using ion gates

A method to control the duty cycle of a time-of-flight mass spectrometer is described. The method relies on one or more ion gates placed in the beam path that have the function to transmit or stop the beam. These ion gates can switch from the open state to the closed state in tens of nanoseconds and effectively select portions of the mass range. The method is useful in circumstances where recording the complete mass spectrum is not an essential requirement, for example, in the analysis of known compounds where sensitivity and speed of operation are more important. It will be of benefit for applications in separation sciences with techniques involving fast chromatographic separations, where hundreds of mass spectra may be required per second. In such circumstances analytical identification may require only a limited number of masses (or mass regions) to be continuously monitored. Improvement of the duty cycle is particularly important for orthogonal-acceleration time-of-flight (oa-TOF) mass spectrometry instruments whose performance suffers from a low duty cycle. The duty cycle is not a constant for an instrument design but is a mass-dependent function and is least for smaller masses. The method described here is capable of raising the duty cycle to 100%. A theory is developed for one or more ion gate arrangements, for both linear- and reflectron-TOF systems. For a two-gate system the relationship between the positions of the first and second gates is described by a '2/3 rule'. Experimental results are shown for one-gate and two-gate operation, both in linear and in reflectron modes of operation, on an oa-TOF system built in-house.     

Spectral counting robust on high mass accuracy mass spectrometers

Mass spectrometry is central to shotgun proteomics, an application that seeks to quantify as much of the total protein complement of a biological sample as possible. The high mass accuracy, resolution, capacity and scan rate of modern mass spectrometers have greatly facilitated this endeavor. The sum of MS to MS/MS transitions in tandem mass spectrometry, the spectral count (SC), of a peptide has been shown to be a reliable estimate of its relative abundance. However, when using SCs, optimal MS configurations are crucial in order to maximize the number of low abundant proteins quantified while keeping the estimates for the highly abundant proteins within the linear dynamic range. In this study, LC/MS/MS analysis was performed using an LTQ‐OrbiTrap on a sample containing many highly abundant proteins. Tuning the LTQ‐OrbiTrap mass spectrometer to minimize redundant MS/MS acquisition and to maximize resolution of the proteome by accurately measured m/z ratios resulted in an appreciable increase in quantified low abundant proteins. An exclusion duration of 90 s and an exclusion width of 10 ppm were found best of those tested. The spectral count of individual proteins was found to be highly reproducible and protein abundance ratios were not affected by the different settings that were applied. We conclude that on a high mass accuracy instrument spectral counting is a robust measure of protein abundance even for samples containing many highly abundant proteins and that tuning dynamic exclusion parameters appreciably improves the number of proteins that can be reliably quantified. Copyright © 2010 John Wiley & Sons, Ltd.     

Mapping the ion kinetic energy in a helium microwave plasma orthogonal acceleration time-of-flight mass spectrometer.

The ion kinetic energy of a helium microwave plasma is studied using an orthogonal acceleration time-of-flight mass spectrometer. The ions produced in the plasma are extracted into the mass spectrometer in an 'off-cone' mode (i.e. the helium plasma plume is off the sampler cone), and enter the repelling zone in the x-direction, which is perpendicular to the flight tube. The information of ion initial kinetic energy was obtained from both theoretical calculations and experimental results. The potential influence of two x-direction steering plates (X-steering plates) on the ion energy and signal intensity was examined. The influence of gas composition on the ion kinetic energy was also investigated. The calculated results show that ions with different m/z have different velocity and kinetic energy when they enter the ion modulation zone, and lighter ions have higher velocity and lower kinetic energy. The experimental results obtained demonstrate that the ion signals of different m/z produced with an 'off-cone' sampling helium microwave plasma show similar trends as calculated with the potential difference of the X-steering plates, revealing their narrow kinetic energy distribution in the x-direction. Under typical operating conditions, the x-direction kinetic energy of ions detected mostly range from about 14.9 eV for (7)Li(+) to 16.8 eV for (208)Pb(+).     

Investigation into the factors affecting accuracy of mass measurements on a time-of-flight mass spectrometer using Design of Experiment

The results of an investigation of the parameters which have the most significant effect on the accuracy of mass measurements on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (q-oaToF) are reported. The influence of eight factors is investigated: ion abundances of reference and analyte compounds, mass difference between analyte and reference compounds, quality of calibration, number of reference acquisitions averaged and TDC (time-to-digital converter) settings (resolution, Np multiplier (number of pushes correction factor), minimum number of points, i.e. minimum acquisition width which defines a peak). To extract the maximum information from as few experiments as possible, a Design of Experiment approach was used. The data will be used as a basis for developing guidance on accurate mass measurement on q-oaToF instruments.     

Dual linear ion trap/orthogonal acceleration time-of-flight mass spectrometer with improved precursor ion selectivity

A new hybrid mass spectrometer based on dual linear ion traps (LITs) and an orthogonal acceleration time-of-flight mass spectrometer (oaTOF), that can achieve MS(n) analysis and high-mass-accuracy detection with high sensitivity, has been developed. Dual-LIT was necessary because, in a single LIT plus oaTOF combination, the LIT pressure favorable for high precursor selectivity in MS(n) analysis (less than 1 mTorr) was far different from an optimum damping pressure (50-100 mTorr) for efficient connection to the TOF mass spectrometer. A dual-LIT solved this problem of inconsistency of the optimum pressures by using the first LIT for MS(n) analysis and the second LIT for collisional damping. This dual-LIT/TOF instrument achieved high-sensitivity MS(n) analysis with high precursor-ion selectivity.     

Determination of the collisionally activated dissociation of a substituted indole by orthogonal acceleration quadrupole time-of-flight mass spectrometry

The use of orthogonal acceleration quadrupole time-of-flight (Q-TOF) mass spectrometry to determine the collisionally activated dissociation (CAD) of a test compound 1-(3-[5-[1,2,4-triazol-4-yl]-1H-indol-3-yl]propyl)-4-(2-[3-fluorophenyl]ethyl)piperazine is described. At unit-mass resolution the identity of many ions is ambiguous because of the complexity of the resulting product ion spectrum. Using the high resolution capabilities of the Q-TOF instrument, exact masses for each fragment were determined. These data were used to infer molecular formulas for each fragment through software interpretation and, by further applying chemical intuition, the majority of ions were fully assigned. Additionally, by utilizing in-source fragmentation at high cone voltage, analyses of second-generation products allowed derivation of a consistent sequential fragmentation pathway. This study clearly demonstrates the power of Q-TOF mass spectrometry to elucidate complex product ion spectra.     

Investigation of the quantitative properties of the quadrupole orthogonal acceleration time-of-flight mass spectrometer with electrospray ionisation using 3,4-methylenedioxymethamphetamine

This paper describes the investigation of the potential of a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF) equipped with an atmospheric pressure ionisation interface for quantitative measurements of small molecules separated by reversed phase liquid chromatography. To this end, the detection limits and linear dynamic range in particular were studied in an LC/MS/MS experiment using 3,4-methylenedioxymethamphetamine standards and 3,4-methylenedioxyethylamphetamine for internal standardisation. In a second phase, the experiment was repeated with real biological extracts (whole blood, serum, and vitreous humour). A calibration for 3,4-methylenedioxymethamphetamine and its metabolite 3,4-methylenedioxyamphetamine was prepared in each of these matrices again using 3,4-methylenedioxyethylamphetamine as internal standard. The resulting quantitative data were compared with those obtained by liquid chromatography with fluorescence detection for the same extracts. The Q-TOF results revealed excellent sensitivity and a linear dynamic range of nearly four decades (2-10 000 pg on-column, r(2) = 0.9998, 1/x weighting). Furthermore, all the calibration curves prepared in biological material were superimposable, LC/MS/MS and LC-fluorescence, and the quantitative results for actual samples compared very favourably. It was concluded that the Q-TOF achieves a linear dynamic range for quantitative LC/MS/MS work exceeding that of fluorescence detection and at much better absolute sensitivity. Copyright 1999 John Wiley & Sons, Ltd.     

Ion dispersion near parallel wire grids in orthogonal acceleration time-of-flight mass spectrometry: predicting the effect of the approach angle on resolution

Ions experience small deflections in the vicinity of grids in accelerators and ion mirrors in time-of-flight (TOF) mass spectrometers. Recent experiments with an orthogonal acceleration (oa) TOF instrument have verified that the effect can significantly degrade resolution when ions approach grids at an angle deviating from 90 degrees. The phenomenon becomes significant only when ions have components of velocity at right angles to the wires of the grids. A model is presented in this study to predict this phenomenon for parallel wire grids. The fractional energy spread of ions (calculated in the static TOF-spectrometer frame of reference) scales directly with the approach angle of ions to the grid (as measured from normal approach). The energy spread also scales with the range of angles that is a consequence of the focusing effect in each gap between the wires of the grid. The equations imply that closely spaced parallel wire grids are best for deployment in oa-TOF systems where non-zero approach angles are unavoidable. Such grids are relatively impractical to manufacture and support but rectangular repeat cell grids with relatively few wires at right angles to the source axis are shown experimentally to introduce minimal energy spread. When these grids are rotated by 90 degrees, the resolution measured in a Q-TOF spectrometer is degraded in approximate agreement with the parallel wire model. A practical implication of this work is that grid transmissions in oa-TOF systems may be significantly increased without loss of resolution. Improvements of approximately 200% (V-mode) and approximately 400% (W-mode) in ion transmission were obtained in this study without compromising resolution. This was achieved with approximately 73% transmission grids and greater potential improvements in transmission are being realised since this study with approximately 89% transmission grids having similar geometry.     

Facile sequencing of oligosaccharides by matrix-assisted laser desorption/ionisation on a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer.

N-Linked oligosaccharide mixtures released from a number of standard glycoproteins were derivatised with 3-acetylamino-6-acetylaminoacridine (AA-Ac) using reductive amination. Analysis of these mixtures using an experimental matrix-assisted laser desorption/ionisation (MALDI) hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer provided detailed information about the mass distribution of the glycan derivatives. Collision-induced dissociation of the singly protonated [M + H](+) ions also gave rise to a number of product ions produced by the sequential cleavage of the glycosidic linkages. As fragmentation of the positively charged species occurred predominantly in one direction, i.e., from the non-reducing end of the glycan to the AA-Ac moiety, a considerable amount of information could be obtained with ease about the sequence in which the sugar residues were attached to one another. This derivatisation procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from proteins separated by gel electrophoresis.     

A fully automated method for accurate mass determination using high-performance liquid chromatography with a quadrupole/orthogonal acceleration time-of-flight mass spectrometer

A generic LC/ESI(+)-oaTOFMS method has been developed for routine automated high accuracy mass determinations of different classes of substances. The system makes use of micro-high-performance liquid chromatography and a hybrid quadrupole/orthogonal acceleration time-of-flight (Q-oaTOF) mass spectrometer. Reproducible and accurate mass measurements were obtained using an electrospray dual sprayer with reserpine as reference compound, introduced into the mass spectrometer alternating with the samples. Experiments were performed to optimize analyte/reference response ratio, statistical algorithm correction setting, and analyte concentration. In these experiments, a clear dependence of the mass measurement error on the analyte/reference response ratio was observed. The dependence of average mass error versus different dead time correction algorithm settings (Np factors) was also explored. In the final automated procedure, verified for a statistically significant set of compounds ( approximately 550) obtained from a medicinal chemistry department, about 70% of the analyzed samples satisfied the acceptance criteria fixed at a maximum error of +/-5 ppm (mass range 150-800 Da).     

Confirmation and identification of the impurities in metolachlor using gas chromatography interfaced with orthogonal acceleration time-of-flight mass spectrometry (GC-oaTOFMS)

The confirmation and identification of the impurities in metolachlor (a herbicde) by using gas chromatography-orthogonal acceleration time of flight mass spectrometry (GC-oaTOFMS) and gas chromatography-quadrupole mass spectrometry (GC-qMS) are described. For the accurate mass measurement can be carried by GC-oaTOFMS, the elemental compositions of molecular and fragment ions in the spectra are suggested. In the experiment the average of mass deviations between the measured and theoretical values was below 2.5 mDa. Finally ten impurities were confirmed and identified. They accounted for 94% of the total impurities by weight.     

Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry

The dissection of specific and nonspecific protein complexes in the gas phase is studied by collisionally activated decomposition. In particular, the gas phase dissection of multiple protonated homodimeric Human Galectin I, E. Coli Glyoxalase I, horse heart cytochrome c, and Hen egg Lysozyme have been investigated. Both the Human Galectin I and E. Coli Glyoxalase I enzymes are biologically active as a dimer, exhibiting molecular weights of approximately 30 kDa. Cytochrome c and Lysozyme are monomers, but may aggregate to some extent at high protein concentrations. The gas phase dissociation of these multiple protonated dimer assemblies does lead to the formation of monomers. The charge distribution over the two concomitant monomers following the dissociation of these multiple protonated dimers is found to be highly dissimilar. There is no evident correlation between the solution phase stability of the dimeric proteins and their gas-phase dissociation pattern. Additionally, in the collisionally activated decomposition spectra diffuse ion signals are observed, which are attributed to monomer ions formed via slow decay of the collisionally activated dimer ions inside the reflectron time-of-flight. Although, the formation of these diffuse metastable ions may complicate the interpretation of collisionally activated decomposition mass spectra, especially when studying noncovalent protein complexes, a simple mathematical equation may be used to reveal their origin and pathway of formation.     

Metabolic profiling of yeast culture using gas chromatography coupled with orthogonal acceleration accurate mass time-of-flight mass spectrometry: Application to biomarker discovery

Yeast and yeast cultures are frequently used as additives in diets of dairy cows. Beneficial effects from the inclusion of yeast culture in diets for dairy mammals have been reported, and the aim of this study was to develop a comprehensive analytical method for the accurate mass identification of the ‘global’ metabolites in order to differentiate a variety of yeasts at varying growth stages (Diamond V XP, Yea-Sacc and Levucell). Microwave-assisted derivatization for metabolic profiling is demonstrated through the analysis of differing yeast samples developed for cattle feed, which include a wide range of metabolites of interest covering a large range of compound classes. Accurate identification of the components was undertaken using GC-oa-ToFMS (gas chromatography-orthogonal acceleration-time-of-flight mass spectrometry), followed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) for data reduction and biomarker discovery. Semi-quantification (fold changes in relative peak areas) was reported for metabolites identified as possible discriminative biomarkers (p-value <0.05, fold change >2), including d-ribose (four fold decrease), myo-inositol (five fold increase), l-phenylalanine (three fold increase), glucopyranoside (two fold increase), fructose (three fold increase) and threitol (three fold increase) respectively.