Ultralong transients enhance sensitivity and resolution in Orbitrap-based single-ion mass spectrometry.

Orbitrap-based charge detection mass spectrometry utilizes single-molecule sensitivity to enable mass analysis of even highly heterogeneous, high-mass macromolecular assemblies. For contemporary Orbitrap instruments, the accessible ion detection (recording) times are maximally ~1-2 s. Here by modifying a data acquisition method on an Orbitrap ultrahigh mass range mass spectrometer, we trapped and monitored individual (single) ions for up to 25 s, resulting in a corresponding and huge improvement in signal-to-noise ratio (×5 compared with 1 s), mass resolution (×25) and accuracy in charge and mass determination of Orbitrap-based charge detection mass spectrometry.

Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.

Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level.

In tandem mass spectrometry (MS2)-based multiplexed quantitative proteomics, the complement reporter ion approaches (TMTc and TMTproC) were developed to eliminate the ratio-compression problem of conventional MS2-level approaches. Resolving all high m/z complement reporter ions (∼6.32 mDa-spaced) requires mass resolution and scan speeds above the performance levels of OrbitrapTM instruments. Therefore, complement reporter ion quantification with TMT/TMTpro reagents is currently limited to 5 out of 11 (TMT) or 9 out of 18 (TMTpro) channels (∼1 Da spaced). We first demonstrate that a FusionTM LumosTM Orbitrap can resolve 6.32 mDa-spaced complement reporter ions with standard acquisition modes extended with 3 s transients. We then implemented a super-resolution mass spectrometry approach using the least-squares fitting (LSF) method for processing Orbitrap transients to achieve shotgun proteomics-compatible scan rates. The LSF performance resolves the 6.32 mDa doublets for all TMTproC channels in the standard mass range with transients as short as ∼108 ms (Orbitrap resolution setting of 50,000 at m/z 200). However, we observe a slight decrease in measurement precision compared to 1 Da spacing with the 108 ms transients. With 256 ms transients (resolution of 120,000 at m/z 200), coefficients of variation are essentially indistinguishable from 1 Da samples. We thus demonstrate the feasibility of highly multiplexed, accurate, and precise shotgun proteomics at the MS2 level.

Fourier transform ion cyclotron resonance mass spectrometry at the true cyclotron frequency.

Ion cyclotron resonance (ICR) cells provide stability and coherence of ion oscillations in crossed electric and magnetic fields over extended periods of time. Using the Fourier transform enables precise measurements of ion oscillation frequencies. These precisely measured frequencies are converted into highly accurate mass-to-charge ratios of the analyte ions by calibration procedures. In terms of resolution and mass accuracy, Fourier transform ICR mass spectrometry (FT-ICR MS) offers the highest performance of any MS technology. This is reflected in its wide range of applications. However, in the most challenging MS application, for example, imaging, enhancements in the mass accuracy of fluctuating ion fluxes are required to continue advancing the field. One approach is to shift the ion signal power into the peak corresponding to the true cyclotron frequency instead of the reduced cyclotron frequency peak. The benefits of measuring the true cyclotron frequency include increased tolerance to electric fields within the ICR cell, which enhances frequency measurement precision. As a result, many attempts to implement this mode of FT-ICR MS operation have occurred. Examples of true cyclotron frequency measurements include detection of magnetron inter-harmonics of the reduced cyclotron frequency (i.e., the sidebands), trapping field-free (i.e., screened) ICR cells, and hyperbolic ICR cells with quadrupolar ion detection. More recently, ICR cells with spatially distributed ion clouds have demonstrated attractive performance characteristics for true cyclotron frequency ion detection. Here, we review the corresponding developments in FT-ICR MS over the past 40 years.

Drosophila melanogaster cloak their eggs with pheromones, which prevents cannibalism.

Oviparous animals across many taxa have evolved diverse strategies that deter egg predation, providing valuable tests of how natural selection mitigates direct fitness loss. Communal egg laying in nonsocial species minimizes egg predation. However, in cannibalistic species, this very behavior facilitates egg predation by conspecifics (cannibalism). Similarly, toxins and aposematic signaling that deter egg predators are often inefficient against resistant conspecifics. Egg cannibalism can be adaptive, wherein cannibals may benefit through reduced competition and added nutrition, but since it reduces Darwinian fitness, the evolution of anticannibalistic strategies is rife. However, such strategies are likely to be nontoxic because deploying toxins against related individuals would reduce inclusive fitness. Here, we report how D. melanogaster use specific hydrocarbons to chemically mask their eggs from cannibal larvae. Using an integrative approach combining behavioral, sensory, and mass spectrometry methods, we demonstrate that maternally provisioned pheromone 7,11-heptacosadiene (7,11-HD) in the eggshell's wax layer deters egg cannibalism. Furthermore, we show that 7,11-HD is nontoxic, can mask underlying substrates (for example, yeast) when coated upon them, and its detection requires pickpocket 23 (ppk23) gene function. Finally, using light and electron microscopy, we demonstrate how maternal pheromones leak-proof the egg, consequently concealing it from conspecific larvae. Our data suggest that semiochemicals possibly subserve in deceptive functions across taxa, especially when predators rely on chemical cues to forage, and stimulate further research on deceptive strategies mediated through nonvisual sensory modules. This study thus highlights how integrative approaches can illuminate our understanding on the adaptive significance of deceptive defenses and the mechanisms through which they operate.

Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade.

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs' structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group's contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb's characterization workflows.

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry.

The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.

Vacuum Laser Photoionization inside the C-trap of an Orbitrap Mass Spectrometer: Resonance-Enhanced Multiphoton Ionization High-Resolution Mass Spectrometry.

State-of-the-art mass spectrometry with ultraviolet (UV) photoionization is mostly limited to time-of-flight (ToF) mass spectrometers with 1000-10 000 m/Δm mass resolution. However, higher resolution and higher spectral dynamic range mass spectrometry may be indispensable in complex mixture characterization. Here, we present the concept, implementation, and initial evaluation of a compact ultrahigh-resolution mass spectrometer with gas-phase laser ionization. The concept is based on direct laser photoionization in the ion accumulation and ejection trap (C-trap) of an Orbitrap mass spectrometer. Resonance-enhanced multiphoton ionization (REMPI) using 266 nm UV pulses from a frequency-quadrupled Nd:YAG laser was applied for selective and efficient ionization of monocyclic and polycyclic aromatic hydrocarbons. The system is equipped with a gas inlet for volatile compounds and a heated gas chromatography coupling. The former can be employed for rapid system m/z-calibration and performance evaluation, whereas the latter enables analysis of semivolatile and higher-molecular-weight compounds. The capability to evaluate complex mixtures is demonstrated for selected petrochemical materials. In these experiments, several hundred to over a thousand compounds could be attributed with a root-mean-square mass error generally below 1 ppm and a mass resolution of over 140 000 at 200 m/z. Isobaric interferences could be resolved, and narrow mass splits, such as 3.4 mDa (SH4/C3), are determined. Single laser shots provided limits of detection in the 20-ppb range for p-xylene and 1,2,4-trimethylbenzene, similar to compact vacuum REMPI-ToF systems.

Imaging of Triglycerides in Tissues Using Nanospray Desorption Electrospray Ionization (Nano-DESI) Mass Spectrometry.

Nonpolar triglycerides (TGs) are rarely detected in mass spectrometry imaging (MSI) experiments unless they are abundant in the sample. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to explore the role of the solvent composition and ionic dopants on the detection of TGs in a murine gastrocnemius muscle tissue used as a model system. We evaluated three solvent mixtures for their ability to extract nonpolar TG species: MeOH:H2O 9:1 (v/v), MeOH:DCM 6:4 (v/v) and MeOH:AcN:tol 5:3.5:1.5 (v/v/v). We observe that TGs are mainly detected as [M+K]+ adducts and their extraction efficiency is improved using less polar solvents: MeOH:DCM and MeOH:AcN:tol. We also explore whether the ionization efficiency of TGs may be improved by doping the MeOH:AcN:tol solvent with ammonium formate (AF) and other ionic additives. However, the formation of [M+NH4]+ adducts of TGs is less efficient than the formation of [M+K]+ adducts in the range of AF concentrations from 0.1 to 10 mM. Chemical derivatization using 100 µM of Girard T reagent predominately generates reaction products of phosphatidylcholine rather than TG species. Moreover, the presence of the Girard T reagent suppresses ion signals of all the species in the spectrum including TGs. Nano-DESI MSI experiments performed using MeOH:AcN:tol solvent enable imaging of TGs without any detectable adverse effect on signals of other lipids and metabolites. Specifically, 10 out of 14 TG species were detected exclusively using MeOH:AcN:tol and the sensitivity towards other TGs was improved by at least an order of magnitude. Although polyunsaturated TGs may be detected using both solvents, saturated and monounsaturated TGs are only detected using MeOH:AcN:tol. Our results provide a direct path for the improved detection of TGs in tissue imaging experiments using liquid-based ambient ionization techniques.

Multiplexed Middle-Down Mass Spectrometry as a Method for Revealing Light and Heavy Chain Connectivity in a Monoclonal Antibody.

Pairing light and heavy chains in monoclonal antibodies (mAbs) using top-down (TD) or middle-down (MD) mass spectrometry (MS) may complement the sequence information on single chains provided by high-throughput genomic sequencing and bottom-up proteomics, favoring the rational selection of drug candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural units that contain the required information on chain pairing. These subunits can be enzymatically produced from whole mAbs and interrogated in their intact form by TD/MD MS approaches. However, the high structural complexity of F(ab) subunits requires increased sensitivity of the modern TD/MD MS for a comprehensive structural analysis. To address this and similar challenges, we developed and applied a multiplexed TD/MD MS workflow based on spectral averaging of tandem mass spectra (MS/MS) across multiple liquid chromatography (LC)-MS/MS runs acquired in reduced or full profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS). We first benchmark the workflow using myoglobin as a reference protein, and then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms of increased signal-to-noise ratio of product ions from utilizing multiple LC-MS/MS runs for TD/MD protein analysis using mass spectral averaging. The workflow performance is compared with the earlier introduced multiplexed TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the latter, we also report on enabling absorption mode FT processing and demonstrate its comparable performance to the enhanced FT (eFT) spectral representation.

Monitoring Membrane Lipidome Turnover by Metabolic 15N Labeling and Shotgun Ultra-High-Resolution Orbitrap Fourier Transform Mass Spectrometry.

Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic 15N labeling and shotgun ultra-high-resolution mass spectrometry (sUHR) to quantify the absolute (molar) abundance and determine the turnover rate of glycerophospholipids and sphingolipids by direct analysis of total lipid extracts. sUHR performed on a commercial Orbitrap Elite instrument at the mass resolution of 1.35 × 106 (m/z 200) baseline resolved peaks of 13C isotopes of unlabeled and monoisotopic peaks of 15N labeled lipids (Δm = 0.0063 Da). Therefore, the rate of metabolic 15N labeling of individual lipid species could be determined without compromising the scope, accuracy, and dynamic range of full-lipidome quantitative shotgun profiling. As a proof of concept, we employed sUHR to determine the lipidome composition and fluxes of 62 nitrogen-containing membrane lipids in human hepatoma HepG2 cells.

Intensity-Independent Noise Filtering in FT MS and FT MS/MS Spectra for Shotgun Lipidomics.

Shotgun lipidomics relies on the direct infusion of total lipid extracts into a high resolution tandem mass spectrometer. A single shotgun analysis produces several hundred of densely populated FT MS and FT MS/MS spectra, each of which might comprise thousands of peaks although a very small percentage of those belong to lipids. Eliminating noise by adjusting a minimal peak intensity threshold is biased and inefficient since lipid species and classes vary in their natural abundance and ionization capacity. We developed a method of peak intensity-independent noise filtering in shotgun FT MS and FT MS/MS spectra that capitalizes on a stable composition of the infused analyte leading to consistent time-independent detection of its bona fide components. Repetition rate filtering relies on a single quantitative measure of peaks detection reproducibility irrespectively of their absolute intensities, masses, or assumed elemental compositions. In comparative experiments, it removed more than 95% of signals detectable in shotgun spectra without compromising the accuracy and scope of lipid identification and quantification. It also accelerated spectra processing by 15-fold and increased the number of simultaneously processed spectra by ∼500-fold hence eliminating the major bottleneck in high-throughput bottom-up shotgun lipidomics.

Atmospheric pressure thermal ionization ion source for peptide analysis.

A novel atmospheric pressure thermal ionization (APTI) ion source was developed for the analysis of liquid samples. The feasibility of the ion source was demonstrated on peptides using two configurations-assisted by hot wire or hot surface. Microalloyed molybdenum, used as a thermal ion- emitter, notably facilitated the formation of multiply-charged ions, but fragmentation products were still observed. Peptide fragmentation accompanying thermal ionization can be used for peptide identification. The described method is promising for studies of biological samples with minimal pre-treatment.

Producing absorption mode Fourier transform ion cyclotron resonance mass spectra with non-quadratic phase correction functions.

RATIONALE: Previously described methods for producing absorption mode Fourier transform ion cyclotron resonance (FTICR) mass spectra have all relied on the phase correction function being quadratic. This assumption has been found to be invalid for some instruments and spectra and so it has not been possible to produce absorption mode spectra for these cases. METHODS: The Autophaser algorithm has been adapted to allow nth order polynomial phase correction functions to be optimized. The data was collected on a modified Thermo LTQ FTICR mass spectrometer, using electrospray ionization and a novel ICR cell design (NADEL). Peak assignment and mass calibration were undertaken using the pyFTMS framework. RESULTS: An nth-order phase correction function has been used to produce an absorption mode mass spectrum of the maltene fraction of a crude oil sample which was not possible using the previous assumption that the phase correction function must be quadratic. Data processing for this spectrum in absorption mode has shown the expected benefits in terms of increasing the number of assigned peaks and also improving the mass accuracy (i.e. confidence) of the assignments. CONCLUSIONS: It is possible to phase-correct time-domain data in FTICRMS to yield absorption mode mass spectra representation even when the data does not correspond to the theoretical quadratic phase correction function predicted by previous studies. This will allow a larger proportion of spectra to be processed in absorption mode.

High-resolution Fourier transform ion cyclotron resonance mass spectrometry with increased throughput for biomolecular analysis.

A multielectrode ion cyclotron resonance (ICR) cell, herein referred to as the "4X cell", for signal detection at the quadruple frequency multiple was implemented and characterized on a commercial 10 T Fourier transform ICR mass spectrometer (FT-ICR MS). Notably, with the 4X cell operating at a 10 T magnetic field we achieved a 4-fold increase in MS acquisition rate per unit of resolving power for signal detection periods typically employed in FTMS, viz., shorter than 6 s. Effectively, the obtained resolution performance represents the limit of the standard measurement principle with dipolar signal detection and FT signal processing at an equivalent magnetic field of 40 T. In other words, the achieved resolving powers are 4 times higher than those provided by 10 T FT-ICR MS with a standard ICR cell. For example, resolving powers of 170,000 and 70,000 were obtained in magnitude-mode Fourier spectra of 768 and 192 ms apodized transient signals acquired for a singly charged fluorinated phosphazine (m/z 1422) and a 19-fold charged myoglobin (MW 16.9 kDa), respectively. In peptide analysis, the baseline-resolved isotopic fine structures were obtained with as short as 768 ms transients. In intact protein analysis, the average resolving power of 340,000 across the baseline-resolved (13)C isotopic pattern of multiply charged ions of bovine serum albumin was obtained with 1.5 s transients. The dynamic range and the mass measurement accuracy of the 4X cell were found to be comparable to the ones obtained for the standard ICR cell on the same mass spectrometer. Overall, the reported results validate the advantages of signal detection at frequency multiples for increased throughput in FT-ICR MS, essential for numerous applications with time constraints, including proteomics.

Self-assembly of a giant molecular Solomon link from 30 subcomponents.

The synthesis of topologically complex structures, such as links and knots, is one of the current challenges in supramolecular chemistry. The so-called Solomon link consists of two doubly interlocked rings. Despite being a rather simple link from a topological point of view, only few molecular versions of this link have been described so far. Here, we report the quantitative synthesis of a giant molecular Solomon link from 30 subcomponents. The highly charged structure is formed by assembly of 12 cis-blocked Pt(2+) complexes, six Cu(+) ions, and 12 rigid N-donor ligands. Each of the two interlocked rings is composed of six repeating Pt(ligand) units, while the six Cu(+) ions connect the two rings. With a molecular weight of nearly 12 kDa and a diameter of 44.2 Å, this complex is the largest non-DNA-based Solomon link described so far. Furthermore, it represents a molecular version of a "stick link".

Adding Color to Mass Spectra of Biopolymers: Charge Determination Analysis (CHARDA) Assigns Charge State to Every Ion Peak.

Traditionally, mass spectrometry (MS) output is the ion abundance plotted versus the ionic mass-to-charge ratio m/z. While employing only commercially available equipment, Charge Determination Analysis (CHARDA) adds a third dimension to MS, estimating for individual peaks their charge states z starting from z = 1 and color coding z in m/z spectra. CHARDA combines the analysis of ion signal decay rates in the time-domain data (transients) in Fourier transform (FT) MS with the interrogation of mass defects (fractional mass) of biopolymers. Being applied to individual isotopic peaks in a complex protein tandem (MS/MS) data set, CHARDA aids peptide mass spectra interpretation by facilitating charge-state deconvolution of large ionic species in crowded regions, estimating z even in the absence of an isotopic distribution (e.g., for monoisotopic mass spectra). CHARDA is fast, robust, and consistent with conventional FTMS and FTMS/MS data acquisition procedures. An effective charge-state resolution Rz ≥ 6 is obtained with the potential for further improvements.

Characterization of the Time-Domain Isotopic Beat Patterns of Monoclonal Antibodies in Fourier Transform Mass Spectrometry.

The time-domain transients in the Fourier transform mass spectrometry (FTMS) analysis of monoclonal antibodies (mAbs) are known to exhibit characteristic isotopic beat patterns. These patterns are defined by the isotopic distributions of all gaseous mAb ions present in the FTMS mass analyzer, originating from single or multiple charge states, and from single or multiple proteoforms. For an isolated charge state of a single proteoform, the mAb isotopic beat pattern resembles narrow splashes of signal amplitude (beats), spaced periodically in the time-domain transient, with broad (often exceeding 1 s) "valleys" between them. Here, we reinforce the importance of isotopic beat patterns for the accurate interpretation and presentation of FTMS data in the analysis of mAbs and other large biopolymers. An updated, mAb-grade version of the transient-mediated FTMS data simulation and visualization tool, FTMS Simulator is introduced and benchmarked. We then apply this tool to evaluate the charge-state dependent characteristics of isotopic beats in mAbs analyses with modern models of Orbitrap and ion cyclotron resonance (ICR) FTMS instruments, including detection of higher-order harmonics. We demonstrate the impact of the isotopic beat patterns on the analytical characteristics of the resulting mass spectra of individual and overlapping mAb proteoforms. The results reported here detail highly nonlinear dependences of resolution and signal-to-noise ratio on the time-domain transient period, absorption or magnitude mode spectra representation, and apodization functions. The provided description and the demonstrated ability to routinely conduct accurate simulations of FTMS data for large biopolymers should aid the end-users of Orbitrap and ICR FTMS instruments in the analysis of mAbs and other biopolymers, including viruses.

Improved Uranium Isotope Ratio Analysis in Liquid Sampling-Atmospheric Pressure Glow Discharge/Orbitrap FTMS Coupling through the Use of an External Data Acquisition System.

Isotope ratio (IR) analysis of natural abundance uranium presents a formidable challenge for mass spectrometry (MS): the required spectral dynamic range needs to enable the quantitatively accurate measurement of the 234UO2 species present at ∼0.0053% isotopic abundance. We address this by empowering a benchtop Orbitrap Fourier transform mass spectrometer (FTMS) coupled with the liquid sampling-atmospheric pressure glow discharge (LS-APGD) ion source and an external high-performance data acquisition system, FTMS Booster X2. The LS-APGD microplasma has demonstrated impressive capabilities regarding elemental and IR analysis when coupled with Orbitrap FTMS. Despite successes, there are limitations regarding the dynamic range and mass resolution that stem from space charge effects and data acquisition and processing restrictions. To overcome these limitations, the FTMS Booster was externally interfaced to an LS-APGD Q Exactive Focus Orbitrap FTMS to obtain time-domain signals (transients) and to process unreduced data. The unreduced time-domain data acquisition with user-controlled processing permit the evaluation of the effects of in-hardware transient phasing, increased transient lengths, advanced transient coadding, varying the length of a transient to be processed with a user-defined time increment, and the use of absorption-mode FT (aFT) processing methods on IR analysis. The added capabilities extend the spectral dynamic range of the instrument to at least 4-5 orders of magnitude and provide a resolution improvement from ∼70k to 900k m/Δm at 200 m/z. The empowered LS-APGD Orbitrap platform allows for the simultaneous measurement of 234UO2 and the prominent 235UO2 and 238UO2 isotopic species at their natural abundances, ultimately yielding improvements in performance when compared to previous uranium IR results on this same Q Exactive Focus instrument.

Transient-Mediated Simulations of FTMS Isotopic Distributions and Mass Spectra to Guide Experiment Design and Data Analysis.

Fourier transform mass spectrometry (FTMS) applications require accurate analysis of extremely complex mixtures of species in wide mass and charge state ranges. To optimize the related FTMS data analysis accuracy, parameters for data acquisition and the allied data processing should be selected rationally, and their influence on the data analysis outcome is to be understood. To facilitate this selection process and to guide the experiment design and data processing workflows, we implemented the underlying algorithms in a software tool with a graphical user interface, FTMS Isotopic Simulator. This tool computes FTMS data via time-domain data (transient) simulations for user-defined molecular species of interest and FTMS instruments, including diverse Orbitrap FTMS models, followed by user-specified FT processing steps. Herein, we describe implementation and benchmarking of this tool for analysis of a wide range of compounds as well as compare simulated and experimentally generated FTMS data. In particular, we discuss the use of this simulation tool for narrowband, broadband, and low- and high-resolution analysis of small molecules, peptides, and proteins, up to the level of their isotopic fine structures. By demonstrating the allied FT processing artifacts, we raise awareness of a proper selection of FT processing parameters for modern applications of FTMS, including intact mass analysis of proteoforms and top-down proteomics. Overall, the described transient-mediated approach to simulate FTMS data has proven useful for supporting contemporary FTMS applications. We also find its utility in fundamental FTMS studies and creating didactic materials for FTMS teaching.