Effect of post-excitation radius on ion abundance, mass measurement accuracy, and isotopic distributions in Fourier transform ion cyclotron resonance mass spectrometry

We report an evaluation of a modern Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) instrument to determine the general trend of post-excitation radius on total ion abundance, mass measurement accuracy, and isotopic distributions for internally calibrated mass spectra. The optimum post-excitation radius was determined using total ion abundance, mass measurement accuracy (MMA), and isotope ratios. However, despite the utility of internal calibration for achieving ultimate MMA, the internal calibrant ions were insufficient for compensating for sub-optimum ICR cell conditions. The findings presented herein underscore the importance of determining the optimal post-excitation radius in FT-ICR-MS to achieve high ion abundance (low limits of detection), high MMA, and valid isotopic distributions.     

Calibration laws based on multiple linear regression applied to matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Operation of any mass spectrometer requires implementation of mass calibration laws to translate experimentally measured physical quantities into a m/z range. While internal calibration in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) offers several attractive features, including exposure of calibrant and analyte ions to identical experimental conditions (e.g. space charge), external calibration affords simpler pulse sequences and higher throughput. The automatic gain control method used in hybrid linear trap quadrupole (LTQ) FT-ICR-MS to consistently obtain the same ion population is not readily amenable to matrix-assisted laser desorption/ionization (MALDI) FT-ICR-MS, due to the heterogeneous nature and poor spot-to-spot reproducibility of MALDI. This can be compensated for by taking external calibration laws into account that consider magnetic and electric fields, as well as relative and total ion abundances. Herein, an evaluation of external mass calibration laws applied to MALDI-FT-ICR-MS is performed to achieve higher mass measurement accuracy (MMA).     

Mass measurement errors caused by “local” frequency perturbations in FTICR mass spectrometry

One of the key qualities of mass spectrometric measurements for biomolecules is the mass measurement accuracy (MMA) obtained. FTICR presently provides the highest MMA over a broad m/z range. However, due to space charge effects, the achievable MMA crucially depends on the number of ions trapped in the ICR cell for a measurement. Thus, beyond some point, as the effective sensitivity and dynamic range of a measurement increase, MMA tends to decrease. While analyzing deviations from the commonly used calibration law in FTICR we have found systematic errors which are not accounted for by a "global" space charge correction approach. The analysis of these errors and their dependence on charge population and post-excite radius have led us to conclude that each ion cloud experiences a different interaction with other ion clouds. We propose a novel calibration function which is shown to provide an improvement in MMA for all the spectra studied.     

Sub parts-per-million mass measurement accuracy of intact proteins and product ions achieved using a dual electrospray ionization quadrupole fourier transform ion cyclotron resonance mass spectrometer

High mass measurement accuracy (MMA) is demonstrated for intact proteins and subsequent collision-induced dissociation product ions using internal calibration. Internal calibration was accomplished using a dual electrospray ionization source coupled with a hybrid quadrupole Fourier transform ion cyclotron resonance (Q-FT-ICR) mass spectrometer. Initially, analyte ions generated via the first electrospray (ESI) emitter are isolated and dissociated in the external quadrupole. This event is followed by a simultaneous switch to the calibrant ion ESI emitter and a disablement of the isolation and activation of the external quadrupole such that a broad m/z range of calibrant ions are accumulated before injecting the analyte/calibrant ion mixture into the ICR cell. Two different internal calibrant solutions were utilized in these studies to evaluate this approach for the top-down characterization of melittin and ubiquitin. While external calibration of protein fragments resulted in absolute MMA greater than 16 ppm, internal standardization significantly improved upon the MMA of both the intact proteins and their products ions which ranged from -2.0 ppm to 1.1 ppm, with an average of -0.9 ppm. This method requires limited modification to ESI-FT-ICR mass spectrometers and is applicable for both positive and negative ionization modes.     

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dual-channel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (<20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ion-energy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10(3), and >10(5) for peptide abundances in the overall separation.     

Automatic gain control in mass spectrometry using a jet disrupter electrode in an electrodynamic ion funnel

We report on the use of a jet disrupter electrode in an electrodynamic ion funnel as an electronic valve to regulate the intensity of the ion beam transmitted through the interface of a mass spectrometer in order to perform automatic gain control (AGC). The ion flux is determined by either directly detecting the ion current on the conductance limiting orifice of the ion funnel or using a short mass spectrometry acquisition. Based upon the ion flux intensity, the voltage of the jet disrupter is adjusted to alter the transmission efficiency of the ion funnel to provide a desired ion population to the mass analyzer. Ion beam regulation by an ion funnel is shown to provide control to within a few percent of a targeted ion intensity or abundance. The utility of ion funnel AGC was evaluated using a protein tryptic digest analyzed with liquid chromatography Fourier transform ion cyclotron resonance (LC-FTICR) mass spectrometry. The ion population in the ICR cell was accurately controlled to selected levels, which improved data quality and provided better mass measurement accuracy.     

The validation of exact mass measurements for small molecules using FT-ICRMS for improved confidence in the selection of elemental formulas

The confidence in an individual measurement is the most important factor when selecting the elemental formula candidates from the list of possible elemental compositions following an exact mass measurement. It is the single mass measurement capability rather than the averaged mass measurement potential of the mass spectrometer that is the critical factor when validating the exact mass measurements of small molecules. Here, an experimental protocol has been established to determine the frequency of exact mass measurement by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS) at known relative ion abundance ratios (RA). This in turn allows for statements about the confidence limit for any single exact mass measurement to be made. This is particularly crucial for a high throughput, automated environment where operator intervention is required to be minimal and repeat analyses are to be avoided. The relative ion abundance calculations are essential to determine the working ranges for specific sample ion abundances. Further, it has been shown that if the sample ion abundance is low, then the ion abundance range for the calibration file does not need to be exactly or closely matched, again benefiting the high throughput application.     

Analysis and elimination of systematic errors originating from coulomb mutual interaction and image charge in Fourier transform ion cyclotron resonance precise mass difference measurements

The effect of mutual Coulomb-mediated interactions between ions of two different mass-to-charge ratios (but equal ion cyclotron orbital radii) on their Fourier transform ion cyclotron resonance (FT/ICR) mass spectral frequency difference is derived analytically and measured experimentally. For a cylindrical ion trap, ion packets are modeled theoretically as infinitely extended lines of charge, and contributions to cyclotron frequency difference due to direct Coulomb repulsion between the lime charges as well as the forces arising from image charge induced on the trap electrodes by each line charge are calculated. A striking theoretical prediction is that the effect on ICR frequency difference of mutual Coulomb repulsion between ions in a mass doublet may be compensated by the image-charge effect. As a result, there is an optimal (calculable) ion cyclotron orbital radius at which the measured cyclotron orbital frequency difference between ions of two different mass-to-charge ratios is independent of mutual Coulomb-mediated interactions between the two components of the mass doublet! Moreover, if the two mass-doublet component ions are present in equal numbers, then the measured ion cyclotron orbital frequency difference is also independent of all Coulomb-mediated interactions between the two types of ions! Thus, the single largest systematic error in measurement of mass difference in a mass doublet by FT/ICR mass spectrometry may be virtually eliminated by appropriate control of ICR orbital radius and/or by performing measurements at various relative abundance ratios and extrapolating to equal relative abundance of the two mass-doublet components. We report experimental tests and verification of these predictions for two different mass doublets: ³He⁺/³H⁺ (cylindrical trap at 4.7 Tesla) and ¹²C¹H ₂ ⁺ /¹⁴ N⁺ (cubic trap at 7.0 Tesla). From the latter measurement, we determine the mass of atomic nitrogen as m(¹⁴N)=14.003 074 014(19) u.     

Improved ion optics for introduction of ions into a 9.4‐T Fourier transform ion cyclotron resonance mass spectrometer

Enhancements to the ion source and transfer optics of our 9.4 T Fourier transform ion cyclotron resonance (ICR) mass spectrometer have resulted in improved ion transmission efficiency for more sensitive mass measurement of complex mixtures at the MS and MS/MS levels. The tube lens/skimmer has been replaced by a dual ion funnel and the following octopole by a quadrupole for reduced ion cloud radial expansion before transmission into a mass‐selective quadrupole. The number of ions that reach the ICR cell is increased by an order of magnitude for the funnel/quadrupole relative to the tube lens/skimmer/octopole. Copyright © 2015 John Wiley & Sons, Ltd.     

Calibration function for the orbitrap FTMS accounting for the space charge effect

Ion storage in an electrostatic trap has been implemented with the introduction of the Orbitrap Fourier transform mass spectrometer (FTMS), which demonstrates performance similar to high-field ion cyclotron resonance MS. High mass spectral characteristics resulted in rapid acceptance of the Orbitrap FTMS for Life Sciences applications. The basics of Orbitrap operation are well documented; however, like in any ion trap MS technology, its performance is limited by interactions between the ion clouds. These interactions result in ion cloud couplings, systematic errors in measured masses, interference between ion clouds of different size yet with close m/z ratios, etc. In this work, we have characterized the space-charge effect on the measured frequency for the Orbitrap FTMS, looking for the possibility to achieve sub-ppm levels of mass measurement accuracy (MMA) for peptides in a wide range of total ion population. As a result of this characterization, we proposed an m/z calibration law for the Orbitrap FTMS that accounts for the total ion population present in the trap during a data acquisition event. Using this law, we were able to achieve a zero-space charge MMA limit of 80 ppb for the commercial Orbitrap FTMS system and sub-ppm level of MMA over a wide range of total ion populations with the automatic gain control values varying from 10 to 10⁷.     

Assigning product ions from complex MS/MS spectra: The importance of mass uncertainty and resolving power

This study offers a unique insight into the mass accuracy and resolving power requirements in MS/MS analyses of complex product ion spectra. In the examples presented here, accurate mass assignments were often difficult because of multiple isobaric interferences and centroid mass shifts. The question then arose whether the resolving power of a medium-resolution quadrupole time-of flight (QqTOF) is sufficient or high-resolution Fourier-transform ion cyclotron resonance (FT-ICR) is required for unambiguous assignments of elemental compositions. For the comparison, two paralytic shellfish poisons (PSP), saxitoxin (STX) and neosaxitoxin (NEO), with molecular weights of 299 and 315 g x mol(-1), respectively, were chosen because of the high peak density in their MS/MS spectra. The assessment of QqTOF collision-induced dissociation spectra and FT-ICR infrared multiphoton dissociation spectra revealed that several intrinsic dissociation pathways leading to isobaric fragment ions could not be resolved with the QqTOF instrument and required FT-ICR to distinguish very close mass differences. The second major source of interferences was M + 1 species originating from coactivated 13C12Cc-1 ion contributions of the protonated molecules of the PSPs. The problem in QqTOF MS results from internal mass calibration when the MH+ ions of analyte and mass calibrant are activated at the same time in the collision or trapping cell. Although FT-ICR MS readily resolved these interfering species, the QqTOF did not provide resolving power >20,000 (full width at half maximum) required to separate most isobaric species. We were able to develop a semi-internal QqTOF calibration technique that activated only the isolated 12C isotope species of the protonated molecules, thus reducing the M + 1 interferences significantly. In terms of overall automated elemental formulas assignment, FT-ICR MS achieved the first formula hit for 100% of the product ions, whereas the QqTOF MS hit rate was only 56 and 65% for STX and NEO product ions, respectively. External mass calibration from commercial FT-ICR and QqTOF instruments gave similar results.     

Suspended trapping gas chromatography I fourier transform mass spectrometry for analysis of complex organic mixtures

The superior sensitivity, dynamic range, and mass measurement accuracy of suspended trapping pulse sequences for gas chromatography combined with Fourier transform mass spectrometry (GC/FTMS) separations of complex organic mixtures is demonstrated. By combining intense ionization conditions with a suspended trapping event prior to detection the working range of the trapped ion cell is increased by 10³. Improved detection limits are shown for the GC/FTMS separation of a peppermint oil, with the suspended trapping total ion chromatogram yielding 28 peaks, compared with 15 with a conventional trapping pulse sequence. A fivefold to fifteenfold improvement in signal-to-noise for suspended trapping measurements is also demonstrated with comparison spectra from separations of an unleaded gasoline sample. Suspended trapping spectra show little mass discrimination when an external ion reservoir is used, and chromatographic peak heights differ from conventional spectra by less than 30% if the initial ion population is within the space charge limit of the cell. Finally, average wide band mass measurement errors for components differing in concentration by several orders of magnitude are improved by a factor of 6 to 20 with suspended trapping compared with conventional trapping. For example, average errors of 8.7 ppm are obtained for a suspended trapping GC/FTMS separation of peppermint oil from a single calibration table in which the analysis is performed in the absence of calibrant.     

Differentiation of lisinopril and its RSS diastereomer by liquid chromatography combined with collision‐induced dissociation mass spectrometry

A simple and sensitive liquid chromatography tandem multiple‐stage mass spectrometry (HPLC/MS/MS) method suitable for bulk lisinopril analysis was developed, by which lisinopril and its RSS isomer were separated and differentiated. In the collision‐induced dissociation (CID) mass spectra of the [M + H]⁺ ions, the abundance of the fragment ion of m/z 246 for lisinopril was about two times higher than the ion of m/z 245; however, the former fragment ion was noted to be a little lower than the latter for RSS isomer at all collision energies. In the CID mass spectra of the [M + Li]⁺ ion, the abundance of the rearrangement ion of m/z 315 for the RSS isomer was about three times higher than that for lisinopril. Furthermore, the difference was supported by the results of energy‐resolved mass spectrometry (ERMS) in the test range of collision energies. Similar differences were also observed between the CID mass spectra of lisinopril and RSS isomer methylester, which indicated that the RSS isomer could be rapidly characterized by the CID mass spectra of both the protonated and lithium adduct ion. Elemental compositions of all the ions were confirmed by Fourier Transform ion cyclotron resonance ESI mass spectrometry (FT‐ICR‐ESI/MS). In addition, theoretical computations were carried out to support the experimental results. Copyright © 2009 John Wiley & Sons, Ltd.     

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

The fundamentals of applying electrospray ionization mass spectrometry to low mass poly(methyl methacrylate) polymers

Electrospray ionization (ESI) is capable of ionizing many soluble polymers. The ESI spectra are complex because of overlap of the multiply charged ions of the oligomer distribution, causing current computer transform programs to fail. However, it is possible to determine the origin of the multiply charged ions, making it feasible to write a program designed to transform ESI polymer spectra. To assess the value of such a program for polymer analysis, isolated monodisperse methyl methacrylate (MMA) oligomers (25 and 50 repeat units) were used to determine molar signal response and propensity for fragmentation. The sum of the peak areas for the multiply charged MMA 50-mer was found to be only about 66% of the summed peak areas for the 25-mer for the same molar concentration. However, conversion of the multiply charged peak areas to the singly charged representations, with peak area compression taken into account, gave equal signal responses for the 25-and 50-mers. Signal response variations due to the tacticity of the MMA oligomers were not observed. Fragmentation of the MMA oligomers also was shown not to occur under normal ESI conditions. Therefore, transformation of the polymer spectra to the singly charged molecular ion distribution should allow accurate calculation of average molecular weights, polydispersity, end group mass, and repeat unit mass.     

A novel fourier transform ion cyclotron resonance mass spectrometer with improved ion trapping and detection capabilities

A novel Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer has been developed for improved biomolecule analysis. A flared metal capillary and an electrodynamic ion funnel were installed in the source region of the instrument for improved ion transmission. The transfer quadrupole is divided into 19 segments, with the capacity for independent control of DC voltage biases for each segment. Restrained ion population transfer (RIPT) is used to transfer ions from the ion accumulation region to the ICR cell. The RIPT ion guide reduces mass discrimination that occurs as a result of time-of-flight effects associated with gated trapping. Increasing the number of applied DC bias voltages from 8 to 18 increases the number of ions that are effectively trapped in the ICR cell. The RIPT ion guide with a novel voltage profile applied during ion transfer provides a 3- to 4-fold increase in the number of ions that are trapped in the ICR cell compared with gated trapping for the same ion accumulation time period. A novel ICR cell was incorporated in the instrument to reduce radial electric field variation for ions with different z-axis oscillation amplitudes. With the ICR cell, called trapping ring electrode cell (TREC), we can tailor the shape of the trapping electric fields to reduce dephasing of coherent cyclotron motion of an excited ion packet. With TREC, nearly an order of magnitude increase in sensitivity is observed. The performance of the instrument with the combination of RIPT, TREC, flared inlet, and ion funnel is presented.     

Laser desorption/ionization fourier transform mass spectrometry of thin films deposited on silicon by plasma polymerization of acetylene

Thin films deposited on silicon substrate by three different methods of plasma polymerization of acetylene were analyzed by direct laser desorption/ionization Fourier transform mass spectrometry. High-resolution mass spectra showed the presence of carbon clusters and hydrocarbon oligomers in different relative abundances. During unipolar and continuous discharge polymerization of acetylene-hydrogen gas mixtures, quadrupole mass spectra of the plasma constituents showed the presence of molecular species with m/z lower than 100 — mainly peaks of C₄H₂ and C₆H₂. Films produced had smooth surfaces and the corresponding LDI-FTMS spectra displayed only carbon cluster signals in the positive ion mode and both hydrocarbon and carbon cluster signals (with much higher relative abundance of carbon cluster signals) in the negative ion mode. Alternatively, during bipolar discharge with either higher acetylene gas flux (>40 cm³/min) or longer deposition times (>10 min), quadrupole mass spectra of the plasma constituents showed signals corresponding to polycyclic aromatic hydrocarbons (PAH) with m/z higher than 100. SEM pictures of the bipolar thin films demonstrated the presence of “flower” structures and nanoparticles developed on the surface. LDI-FTMS spectra of such thin films showed either total absence or lower relative abundance of carbon cluster signals, compared with hydrocarbon signals.     

Fourier transform ion cyclotron resonance mass spectrometry in a high homogeneity 25 tesla resistive magnet

Many performance parameters of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry improve dramatically with increasing magnetic field. Our prior results from a 20 tesla resistive magnet showed that performance was limited by the large spatial inhomogeneity in spite of the high field. In this paper, we compare matrix-assisted laser desorption/ionization (MALDI) mass spectra at the same magnetic field for two resistive magnets with different field spatial homogeneity. In addition, we report MALDI spectra at 25 tesla—the highest magnetic field for FT-ICR to date. The first broadband FT-ICR mass spectrum [poly(ethylene glycol) 2000] from a resistive magnet is accurately fitted by the standard ICR mass calibration function.     

Analysis of multicomponent mass spectra applying Bayesian probability theory

In this paper we develop a method for the decomposition of mass spectra of gas mixtures, together with the relevant calibration measurements. The method is based on Bayesian probability theory. Given a set of spectra, the algorithm returns the relative concentrations and the associated margin of confidence for each component of the mixture. In addition to the concentrations, such a data set enables the derivation of improved values of the cracking coefficients of all contributing species, even for those components for which the set does not contain a calibration measurement. This latter feature also allows one to analyze mixtures that contain radicals in addition to stable molecules. As an example, we analyze and discuss the mass spectra obtained from the pyrolysis of azomethane, which contain the radical CH3 apart from nitrogen and C1- and C2-hydrocarbons.     

TOF-SIMS: Accurate mass scale calibration

A study is presented of the factors affecting the calibration of the mass scale in time-of-flight secondary ion mass spectrometry (TOF-SIMS). At the present time, TOF-SIMS analysts using local calibration procedures achieve a rather poor relative mass accuracy of only 150 ppm for large molecules (647 u) whereas for smaller fragments of <200 u this figure only improves to 60 ppm. The instrumental stability is 1 ppm and better than 10 ppm is necessary for unique identification of species. The above experimental uncertainty can lead to unnecessary confusion where peaks are wrongly identified or peaks are ambiguously assigned. Here we study, in detail, the instrumental parameters of a popular single stage reflection TOF-SIMS instrument with ion trajectory calculations using SIMION. The effect of the ion kinetic energy, emission angle, and other instrumental operating parameters on the measured peak position are determined. This shows clearly why molecular and atomic ions have different relative peak positions and the need for an aperture to restrict ions at large emission angles. These data provide the basis for a coherent procedure for optimizing the settings for accurate mass calibration and rules by which calibrations for inorganics and organics may be incorporated. This leads to a new generic set of ions for mass calibration that improves the mass accuracy in our interlaboratory study by a factor of 5. A calibration protocol is developed, which gives a relative mass accuracy of better than 10 ppm for masses up to 140 u. The effects of extrapolation beyond the calibration range are discussed and a recommended procedure is given to ensure that accurate mass is achieved within a selectable uncertainty for large molecules. Additionally, we can alternatively operate our instrument in a regime with good energy discrimination (i.e., poor energy compensation) to study the fragmented energies of molecules. This leads to data that support previous concepts developed in G-SIMS.