Metabolite identification in metabolomics samples is a key step that critically impacts downstream analysis. We recently introduced the SUMMIT NMR/mass spectrometry (MS) hybrid approach for the identification of the molecular structure of unknown metabolites based on the combination of NMR, MS, and combinatorial cheminformatics. Here, we demonstrate the feasibility of the approach for an untargeted analysis of both a model mixture and E. coli cell lysate based on 2D/3D NMR experiments in combination with Fourier transform ion cyclotron resonance MS and MS/MS data. For 19 of the 25 model metabolites, SUMMIT yielded complete structures that matched those in the mixture independent of database information. Of those, seven top-ranked structures matched those in the mixture, and four of those were further validated by positive ion MS/MS. For five metabolites, not part of the 19 metabolites, correct molecular structural motifs could be identified. For E. coli, SUMMIT MS/NMR identified 20 previously known metabolites with three or more 1H spins independent of database information. Moreover, for 15 unknown metabolites, molecular structural fragments were determined consistent with their spin systems and chemical shifts. By providing structural information for entire metabolites or molecular fragments, SUMMIT MS/NMR greatly assists the targeted or untargeted analysis of complex mixtures of unknown compounds.Keywords: 3D NMR HSQC-TOCSY; COLMAR database; metabolomics; NMR-MS hybrid approach; unknown metabolite identification;

Corrosion control at refineries remains a challenge because the mechanism of naphthenic acid (NAP) corrosion is still not fully understood. The rate of NAP corrosion does not correlate with acidity (as measured by total acid number); therefore, it has been suggested that a subset of NAP in petroleum fractions may be more corrosive than others. Because the primary corrosion product (iron naphthenates) may thermally decompose to ketones at corrosion temperatures (250–400 °C), ketones in corrosion fluids could potentially be used to implicate specific problematic acids in corrosion tests. To that end, we have developed a method for isolating and characterizing ketones in corrosion test solutions. Ketones from tests on palmitic and 4-cyclohexyl pentanoic acids (C16H32O2 and C11H20O2) have been successfully isolated with a strong anion exchange solid-phase separation. Gas chromatography/mass spectrometry identifies ketones formed as a result of model acid corrosion. Fourier transform ion cyclotron resonance mass spectrometry further confirms the detection of these ketones and structurally confirms ketones by use of a commercially available reagent that targets ketones and aldehydes. Additional oxygen species generated in the corrosion test likely result from reactions between dissolved atmospheric oxygen and the mineral oil matrix. With this method now validated, it can be applied in future studies of more complex acid mixtures to determine any structural specificity in naphthenic acid corrosion.

Here, we present atmospheric pressure photoionization (APPI) Fourier transform ion cyclotron resonance (FTICR) mass analysis of a volcanic asphalt sample by acquiring data for 20 Da wide mass segments across a 1000 Da range, stitched into a single composite mass spectrum, and compare to a broad-band mass spectrum for the same sample. The segmented spectrum contained 170 000 peaks with magnitude greater than 6σ of the root-mean-square (rms) baseline noise, for which 126 264 unique elemental compositions could be assigned. Approximately two-thirds of those compositions represent monoisotopic (i.e., chemically different) species. That complexity is higher than that for any previously reported mass spectrum and almost 3 times greater than that obtained from the corresponding broad-band spectrum (59 015). For the segmented mass spectrum, the signal-to-noise ratio (S/N) was significantly higher throughout the spectrum, but especially at the lower and upper ends of mass distribution relative to that of the near-Gaussian broad-band mass distribution. Despite this S/N improvement, mass measurement accuracy was noticeably improved only at lower masses. The increased S/N did, however, yield a higher number of peaks and higher dynamic range throughout the entire segmented spectrum relative to the conventional broad-band spectrum. The additional assigned peaks include higher heteroatom species, as well as additional radicals and isotopologues. Segmenting can require a significant investment in data acquisition and analysis time over broad-band spectroscopy (∼1775% in this case) making it best suited for targeted analysis and/or when complete compositional coverage is important. Finally, the present segmented spectrum contains, to our knowledge, more assigned peaks than any spectrum of any kind (e.g., UV–vis, infrared, microwave, magnetic resonance, etc.).

Waste material pyrolysis has proven useful for the production of pyrolysis oils; however, the physical properties and chemical composition of pyrolysis oils are greatly influenced by the feedstock. It is well established that lignin- and cellulose-rich material produces pyrolysis oils high in aromatic oxygen-containing compounds, whereas pyrolysis oils produced from other sources such as plastics and household wastes are far less characterized. Here, three fast pyrolysis oils produced from landfill waste, recycled plastics, and pine forestry residue are compared by elemental analysis, Fourier transform infrared spectroscopy (FT-IR), comprehensive 2D gas chromatography (GC×GC), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), and liquid chromatography. GC×GC, FT-ICR MS, and liquid chromatography provide insight into the chemical composition of pyrolysis oils, whereas FT-IR analysis identifies functional groups. Landfill and plastic pyrolysis oils were found to contain higher hydrocarbon content that resulted from little or no cellulosic material in their feedstock. In contrast, pine pyrolysis oil is more aromatic and contains a higher abundance of polar species due to the number of oxygen functionalities. The hydrocarbons in plastic pyrolysis oil are more saturated than in landfill and pine pyrolysis oils. Due to their lower oxygen content, landfill and plastic pyrolysis oils are more attractive than pine pyrolysis oil as potential fuel candidates.

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2–0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

It is known that the ion collision cross section (CCS) may be calculated from the linewidth of a Fourier transform ion cyclotron resonance (FT-ICR) mass spectral peak at elevated pressure (e.g., ∼10−6 Torr). However, the high mass resolution of FT-ICR is sacrificed in those experiments due to high buffer gas pressure. In this study, we describe a linewidth correction method to eliminate the windowing-induced peak broadening effect. Together with the energetic ion–neutral collision model previously developed by our group, this method enables the extraction of CCSs of biomolecules from high-resolution FT-ICR mass spectral linewidths, obtained at a typical operating buffer gas pressure of modern FT-ICR instruments (∼10−10 Torr). CCS values of peptides including MRFA, angiotensin I, and bradykinin measured by the proposed method agree well with ion mobility measurements, and the unfolding of protein ions (ubiquitin) at higher charge states is also observed.

Algae lipids contain long-chain saturated and polyunsaturated fatty acids. The lipids may be transesterified to generate biodiesel fuel. Here, we compare polar lipid compositions for two microalgae, Nannochloropsis oculata and Haematococcus pluvialis, that are prospective lipid-rich feedstock candidates for an emerging biodiesel industry. Online nano liquid chromatography coupled with negative electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry ((−)ESI FT-ICR MS) with newly modified ion optics provides ultrahigh mass accuracy and resolving power to identify hundreds of unique elemental compositions. Assignments are confirmed by isotopic fine structure for a polar lipid extract. Collision-induced-dissociation (CID) MS/MS provides additional structural information. H. pluvialis exhibits more highly polyunsaturated lipids than does N. oculata.

This paper summarizes the development of a new method for screening petroleum crude oils for the presence of “ARN” tetraprotic acids, based on molecularly imprinted polymers (MIPs). When slurried with crude oils, MIPs imprinted by a suite of ARN acid standards selectively bind to ARN acids. The strong interaction enables removal of most of the crude oil by several toluene washes. The ARN acids are then recovered by washing with a mixture of 5% formic acid in methanol/toluene. Our results show that the sample load directly impacts the amount of ARN acids recovered: a higher sample load lowers the detectable concentration range. MIPs offer a quick and sensitive method to screen crude oils for ARN acid present at low concentrations (sub-parts per million). This study also illustrates the capabilities of MIPs for isolating members of an entire class of compounds.

To understand the role and function of a biomolecule in a biosystem, it is important to know both its composition and structure. Here, a mass spectrometric based approach has been proposed and applied to demonstrate that collision cross sections and high-resolution mass spectra of biomolecule ions may be obtained simultaneously by Fourier transform ion cyclotron resonance mass spectrometry. With this method, the unfolding phenomena for ubiquitin ions that possess different number of charges have been investigated, and results agree well with ion mobility measurements. In the present approach, we extend ion collision cross-section measurements to lower pressures than in prior ion cyclotron resonance (ICR)-based experiments, thereby maintaining the potentially high resolution of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), and enabling collision cross section (CCS) measurements for high-mass biomolecules.

In the first use of high-field electron paramagnetic resonance (EPR) spectroscopy to characterize paramagnetic metal–organic and free radical species from tar balls and weathered crude oil samples from the Gulf of Mexico (collected after the Deepwater Horizon oil spill) and an asphalt volcano sample collected off the coast of Santa Barbara, CA, we are able to identify for the first time the various paramagnetic species present in the native state of these samples and understand their molecular structures and bonding. The two tar ball and one asphalt volcano samples contain three distinct paramagnetic species: (i) an organic free radical, (ii) a [VO]2+ containing porphyrin, and (iii) a Mn2+ containing complex. The organic free radical was found to have a disc-shaped or flat structure, based on its axially symmetric spectrum. The characteristic spectral features of the vanadyl species closely resemble those of pure vanadyl porphyrin; hence, its nuclear framework around the vanadyl ion must be similar to that of vanadyl octaethyl porphyrin (VOOEP). The Mn2+ ion, essentially undetected by low-field EPR, yields a high-field EPR spectrum with well-resolved hyperfine features devoid of zero-field splitting, characteristic of tetrahedral or octahedral Mn–O bonding. Although the lower-field EPR signals from the organic free radicals in fossil fuel samples have been investigated over the last 5 decades, the observed signal was featureless. In contrast, high-field EPR (up to 240 GHz) reveals that the species is a disc-shaped hydrocarbon molecule in which the unpaired electron is extensively delocalized. We envisage that the measured g-value components will serve as a sensitive basis for electronic structure calculations. High-field electron nuclear double resonance experiments should provide an accurate picture of the spin density distribution for both the vanadyl-porphyrin and Mn2+ complexes, as well as the organic free radical, and will be the focus of follow-up studies.

•History of the development of FT-ICR MS, from conception to date.•Parallels between FT-NMR and FT-ICR MS.•Explanation of multiplex vs. multichannel spectroscopy.This article reviews the development of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in several respects: (a) a strong static magnetic field serves to convert ion mass-to-charge ratio into cyclotron frequency. Because frequency is the most accurately measurable property, ICR MS inherently offers higher mass resolution and mass accuracy than any other mass analyzer. (b) Coherent excitation followed by induced charge detection yields a time-domain signal whose discrete Fourier transform produces a mass spectrum of ions spanning a wide m/z range simultaneously. By simple analogy to weighing objects with a mechanical balance, that “multiplex” advantage can be shown to be equivalent to “multichannel” detection by an array of individual single-channel detectors. (c) FT-ICR MS performance benefits from near-elimination of magnetic field inhomogeneity by inherent ion cyclotron rotation and ion axial oscillation; inherent nearly quadrupolar electrostatic trapping potential and nearly uniform rf electric field homogeneity near the center of the ICR ion trap (both improved even further by recent ICR cell designs); and theoretically optimal excitation and mass selection produced by stored-waveform inverse Fourier transformation (SWIFT). (d) External ion accumulation allows efficient coupling of atmospheric pressure continuous ionization sources (notably electrospray ionization) with pulsed high-vacuum FT-ICR MS excitation/detection, and injection of externally trapped ions through the “magnetic mirror” into the ICR ion trap has been optimized based on ion trajectory simulations. (e) MS/MS can be performed either inside (e.g., electron capture dissociation, infrared multiphoton dissociation) or outside (e.g., collision-induced dissociation, electron transfer dissociation) the ICR ion trap. (f) Finally, FT-ICR MS instrumentation and experimental event sequences have benefited from striking parallels to prior nuclear magnetic resonance spectroscopy developments. Similarly, non-ICR FT MS development (notably the orbitrap) has benefited from FT-ICR precedents.

•First combination of a commercial direct analysis in real time (DART) source with FT-ICR MS.•Abundant molecular or quasimolecular ions from C60, heavy petroleum, naphthenate deposits, and biotar, without fragmentation.•Desorption/ionization of compounds with boiling points significantly higher than the DART source temperature.We report the first combination of a commercial direct analysis in real time (DART) source with FT-ICR MS and its application to analysis of complex organic mixtures. DART enables ionization of compounds with little or no sample preparation, and FT-ICR provides ultrahigh mass resolution and mass accuracy. The combination provides a rapid, robust, and reliable method for analysis of components spanning a wide range of chemical functionality. DART 9.4 T FT-ICR MS generates abundant molecular or quasimolecular ions from C60, heavy petroleum, naphthenate deposits, and biotar, without fragmentation. Moreover, we demonstrate desorption/ionization of compounds with boiling points significantly higher than the DART source temperature. DART FT-ICR MS thus offers a new and useful atmospheric pressure ionization mass spectrometry technique for analysis of complex organic mixtures.

We present laser desorption atmospheric pressure photochemical ionization mass spectrometry (LD/APPCI MS) for rapid throughput analysis of complex organic mixtures, without the need for matrix, electric discharge, secondary electrospray, or solvents/vaporizers. Analytes dried on a microscope slide are vaporized in transmission geometry by a laser beam aligned with the atmospheric pressure inlet of the mass spectrometer. The laser beam initiates a cascade of reactions in the region between the glass slide and MS inlet, leading to generation of reagent ions for chemical ionization of vaporized analyte. Positive analyte ions are generated predominantly by proton transfer, charge exchange, and hydride abstraction, whereas negative ions are generated by electron capture or proton transfer reactions, enabling simultaneous analysis of saturated, unsaturated, and heteroatom-containing hydrocarbons. The absence of matrix interference renders LD/APPCI MS particularly useful for analysis of small molecules (<2000 Da) such as those present in petroleum crude oil and petroleum deposits. [M + H]+ and M+• dominate the positive-ion mass spectra for olefins and polyaromatic hydrocarbons, whereas saturated hydrocarbons are observed mainly as [M – H]+ and/or M+•. Heteroatom-containing hydrocarbons are observed predominantly as [M + H]+. [M – H]− and M–• are the dominant negative ions observed for analytes of lower gas-phase basicity or higher electron affinity than O2. The source was coupled with a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) to resolve and identify thousands of peaks from Athabasca bitumen heavy vacuum gas oil distillates (400–425 and 500–538 °C), enabling simultaneous characterization of their polar and nonpolar composition. We also applied LD/APPCI FTICR MS for rapid analysis of sodium and calcium naphthenate deposits with little to no sample pretreatment to provide mass spectral fingerprints that enable reliable compositional characterization.

Lithium cationization can significantly extend the compositional range for analysis of petroleum components by positive electrospray ionization [(+) ESI], by accessing species that lack a basic nitrogen atom and, hence, are not seen by conventional (+) ESI that relies on protonation as the primary ionization mechanism. Here, various solvent compositions and lithium salts enabled us to optimize ionization by formation of lithium adducts ([M + Li]+), and the results are compared to production of [M + H]+ by conventional (+) ESI with formic acid. Lithium cationization (+) ESI Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) of Athabasca bitumen heavy vacuum gas oil (475–500 °C) and North and South American crude oils demonstrates considerable improvement over protonation for production of ions from compounds belonging to SxOy (SO, SO2, SO3, SO4, S2O, S2O2, etc.) heteroatom classes. Those compounds exhibit much higher affinity for lithium cation than for proton and yield abundant [M + Li]+ ions. Li+ cationization thus opens a pathway for detection and characterization of SxOy class compounds that preferentially concentrate at the interface in oil/water emulsions.

Silver cationization constitutes a complementary approach for analysis of petroleum components with positive-ion electrospray ionization (ESI) mass spectrometry and accesses species that lack a basic nitrogen atom and, hence, are not observed by conventional positive ESI. Four samples of different origin [Canadian bitumen, Canadian bitumen heavy vacuum gas oil (HVGO; 475–500 °C) and South American and Middle East heavy crude oils, all high in sulfur content] were used to study silver cationization by (+) ESI. Cationization with Ag+ is essentially instantaneous and accesses hydrocarbons and nonpolar sulfur-containing heteroatom classes (e.g., Ss and SsOo), providing an attractive alternative to time-consuming derivatization by S-methylation to ionize sulfur-containing species. For each sample, we compare Ag+ cationization (+) ESI to conventional (+) ESI with formic acid to promote ion formation. Other ionization methods, such as chemical ionization (CI), field desorption (FD), matrix-assisted laser desorption ionization (MALDI) chemical ionization, field desorption ionization, and MALDI, are low in throughput and/or involve thermal processes that may degrade substrate molecules from non-volatile high-boiling petroleum components. Mix-and-spray Ag+ cationization avoids tedious separation and time-consuming derivatization and results in the rapid speciation of sulfur-containing compounds in petroleum and its fractions without the need for thermal desorption.

Ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) enables the direct characterization of complex mixtures without prior fractionation. High mass resolution can distinguish peaks separated by as little as 1.1 mDa), and high mass accuracy enables assignment of elemental compositions in mixtures that contain tens of thousands of individual components (crude oil). Negative electrospray ionization (ESI) is particularly useful for the speciation of the most acidic petroleum components that are implicated in oil production and processing problems. Here, we replace conventional ammonium hydroxide by tetramethylammonium hydroxide (TMAH, a much stronger base, with higher solubility in toluene) to more uniformly deprotonate acidic components of complex mixtures by negative ESI FTICR MS. The detailed compositional analysis of four crude oils (light to heavy, from different geographical locations) reveals that TMAH reagent accesses 1.5–6 times as many elemental compositions, spanning a much wider range of chemical classes than does NH4OH. For example, TMAH reagent produces abundant negative electrosprayed ions from less acidic and neutral species that are in low abundance or absent with NH4OH reagent. More importantly, the increased compositional coverage of TMAH-modified solvent systems maintains, or even surpasses, the compositional information for the most acidic species. The method is not limited to petroleum-derived materials and could be applied to the analysis of dissolved organic matter, coal, lipids, and other naturally occurring compositionally complex organic mixtures.

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ∼148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains CH2 and CH3, and the disulfide bond (S–S)-linked heavy-chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S–S spans the polypeptide backbone. Only a few cleavages in the S–S-linked light-chain constant domain, hinge region, and heavy-chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers.

We report the reliable determination of equilibrium protein disulfide bond reduction potentials (E°′) by isotope-coded cysteine alkylation coupled with top-down Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). This technique enables multiple redox-active sites to be characterized simultaneously and unambiguously without the need for proteolysis or site-directed mutagenesis. Our model system was E. coli thioredoxin, and we determined E°′ for its CGPC active-site disulfide as −280 mV in accord with literature values. E°′ for the homologous disulfide in human thioredoxin 1 (Trx1) was determined as −281 mV, a value considerably more negative than the previously reported −230 mV. We also observed S-glutathionylation of Trx1 and localized that redox modification to Cys72; E°′ for the intermolecular disulfide was determined as −186 mV. Intriguingly, that value corresponds to the intracellular glutathione/glutathione disulfide (GSH/GSSG) potential at the redox boundary between cellular differentiation and apoptosis.

Molecular characterization of asphaltenes by conventional analytical techniques is a challenge because of their compositional complexity, high heteroatom content, and asphaltene aggregate formation at low concentrations. Thus, most common characterization techniques rely on bulk properties or solution-phase behavior (solubility). Proposed over 20 years ago, the Boduszynski model proposes a continuous progression in petroleum composition (molecular weight, structure, and heteroatom content) as a function of the atmospheric equivalent boiling point. Although exhaustive detailed compositional analysis of petroleum distillates validates the continuum model, the available compositional data from asphaltene fractions supports the extension of the continuum model into the nondistillables only indirectly. Asphaltenes, defined by their insolubility in alkane solvents, accumulate in high-boiling fractions and form stable aggregate structures at low parts per billion (ppb) concentrations, far below the concentration required for most mass analyzers. Here, we present direct mass spectral detection of stable asphaltene aggregates at lower concentrations than previously published and observe the onset of asphaltene nanoaggregate formation by time-of-flight mass spectrometry (TOF–MS). We conclude that a fraction of asphaltenes must be present as nanoaggregates (not monomers) in all atmospheric pressure and laser-based ionization methods. Thus, those methods access a subset of the asphaltene continuum.

Asphaltenes and maltenes are defined operationally by solubility (in, e.g., heptane). Asphaltenes self-associate in solution and form putative nanoaggregates composed of approximately 6–10 asphaltene monomers per subunit. Bulk measurements indicate that asphaltenes are more aromatic than maltenes and contain more heteroatoms and metals (nitrogen, sulfur, oxygen, nickel, and vanadium). Numerous direct imaging, molecular diffusion, and mass spectral results agree that asphaltenes and maltenes are defined by similar, overlapped carbon number ranges, drastically restricting the acceptable carbon number and aromaticity “compositional space” for asphaltene compounds. Thus, when viewed by a plot of aromaticity versus carbon number for a given heteroatom class, asphaltenes must occupy different compositional space than maltenes because they share the same carbon number range but differ in bulk aromaticity and solution phase behavior. Boduszynski’s work supported overlapping asphaltene/maltene molecular weights, and he proposed that “high boiling does not necessitate high molecular weight” [Boduszynski, M. M.Composition of heavy petroleums. 1. Molecular weight, hydrogen deficiency, and heteroatom concentration as a function of atmospheric equivalent boiling-point up to 1400 °F (760 °C). Energy Fuels 1987, 1 (1) 2−11]. However, his limited mass spectral resolution precluded direct molecular-level confirmation. Current mass spectral results combined with results published in parts 1 (10.1021/ef100149n), 2 (10.1021/ef1001502), and 3 (10.1021/ef3018578) of this series provide the basis for a continuum in petroleum structure and composition in support of the Boduszynski model and confirm that asphaltene molecules share the same carbon number range with their maltene counterparts but are simply more aromatic. Thus, the compositional space for maltenic and monomeric asphaltene species is now known. Part 3 (10.1021/ef3018578) provided evidence for asphaltene aggregate formation at concentrations below that required for most mass spectral analyses, suggesting that, at these concentrations, the majority of asphaltenes are locked in aggregate structures and, therefore, undetected as monomers. Here, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) results confirm that asphaltenes and maltenes of the same heteroatom class exhibit higher aromaticity than maltenes of the same carbon number, limited by the highest possible aromaticity for a stable planar aromatic structure, and clearly differentiate asphaltene and maltene monomeric molecular compositions.

Fourier transform mass spectrometry (FTMS) of the isolated isotopic distribution for a highly charged biomolecule produces time-domain signal containing large amplitude signal “beats” separated by extended periods of much lower signal magnitude. Signal-to-noise ratio for data sampled between beats is low because of destructive interference of the signals induced by members of the isotopic distribution. Selective blanking of the data between beats has been used to increase spectral signal-to-noise ratio. However, blanking also eliminates signal components and, thus, can potentially distort the resulting FT spectrum. Here, we simulate the time-domain signal from a truncated isotopic distribution for a single charge state of an antibody. Comparison of the FT spectra produced with or without blanking and with or without added noise clearly show that blanking does not improve mass accuracy and introduces spurious peaks at both ends of the isotopic distribution (thereby making it more difficult to identify posttranslational modifications and/or adducts). Although the artifacts are reduced by use of multiple Gaussian (rather than square wave) windowing, blanking appears to offer no advantages for identifying true peaks or for mass measurement.

Elemental composition assignment confidence in mass spectrometry is typically assessed by monoisotopic mass accuracy. For a given mass accuracy, resolution and detection of other isotopologues can further narrow the number of possible elemental compositions. However, such measurements require ultrahigh resolving power and high dynamic range, particularly for compounds containing low numbers of nitrogen and oxygen (both 15N and 18O occur at less than 0.4 % natural abundance). Here, we demonstrate validation of molecular formula assignment from isotopic fine structure, based on ultrahigh resolution broadband Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Dynamic range is enhanced by external quadrupole and internal stored waveform inverse Fourier transform (SWIFT) isolation to facilitate detection of low abundance heavy atom isotopologues.

Carbonaceous presolar grains of supernovae origin have long been isolated and are determined to be the carrier of anomalous 22Ne in ancient meteorites. That exotic 22Ne is, in fact, the decay isotope of relatively short-lived 22Na formed by explosive nucleosynthesis, and therefore, a selective and rapid Na physical trapping mechanism must take place during carbon condensation in supernova ejecta. Elucidation of the processes that trap Na and produce large carbon molecules should yield insight into carbon stardust enrichment and formation. Herein, we demonstrate that Na effectively nucleates formation of Na@C60 and other metallofullerenes during carbon condensation under highly energetic conditions in oxygen- and hydrogen-rich environments. Thus, fundamental carbon chemistry that leads to trapping of Na is revealed, and should be directly applicable to gas-phase chemistry involving stellar environments, such as supernova ejecta. The results indicate that, in addition to empty fullerenes, metallofullerenes should be constituents of stellar/circumstellar and interstellar space. In addition, gas-phase reactions of fullerenes with polycyclic aromatic hydrocarbons are investigated to probe “build-up” and formation of carbon stardust, and provide insight into fullerene astrochemistry.

We present the first coupling of laser spray ionization inlet (LSII) and matrix assisted ionization inlet (MAII) to high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) for generation of electrospray-like ions to take advantage of increased sensitivity, mass range, and mass resolving power afforded by multiple charging. We apply the technique to top-down protein analysis and characterization of metalloproteins. We also present a novel method for generation of multiply-charged copper–peptide complexes with varying degrees of copper adduction by LSII. We show an application of the generated copper–peptide complexes for protein charge state and molecular weight determination, particularly useful for an instrument such as a linear ion trap mass analyzer.

The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

The smallest fullerene to form in condensing carbon vapor has received considerable interest since the discovery of Buckminsterfullerene, C60. Smaller fullerenes remain a largely unexplored class of all-carbon molecules that are predicted to exhibit fascinating properties due to the large degree of curvature and resulting highly pyramidalized carbon atoms in their structures. However, that curvature also renders the smallest fullerenes highly reactive, making them difficult to detect experimentally. Gas-phase attempts to investigate the smallest fullerene by stabilization through cage encapsulation of a metal have been hindered by the complexity of mass spectra that result from vaporization experiments which include non-fullerene clusters, empty cages, and metallofullerenes. We use high-resolution FT-ICR mass spectrometry to overcome that problem and investigate formation of the smallest fullerene by use of a pulsed laser vaporization cluster source. Here, we report that the C28 fullerene stabilized by encapsulation with an appropriate metal forms directly from carbon vapor as the smallest fullerene under our conditions. Its stabilization is investigated, and we show that M@C28 is formed by a bottom-up growth mechanism and is a precursor to larger metallofullerenes. In fact, it appears that the encapsulating metal species may catalyze or nucleate endohedral fullerene formation.

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ∼70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

We present atmospheric pressure laser-induced acoustic desorption chemical ionization (AP/LIAD-CI) with O2 carrier/reagent gas as a powerful new approach for the analysis of saturated hydrocarbon mixtures. Nonthermal sample vaporization with subsequent chemical ionization generates abundant ion signals for straight-chain, branched, and cycloalkanes with minimal or no fragmentation. [M – H]+ is the dominant species for straight-chain and branched alkanes. For cycloalkanes, M+• species dominate the mass spectrum at lower capillary temperature (<100 °C) and [M – H]+ at higher temperature (>200 °C). The mass spectrum for a straight-chain alkane mixture (C21–C40) shows comparable ionization efficiency for all components. AP/LIAD-CI produces molecular weight distributions similar to those for gel permeation chromatography for polyethylene polymers, Polywax 500 and Polywax 655. Coupling of the technique to Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for the analysis of complex hydrocarbon mixtures provides unparalleled mass resolution and accuracy to facilitate unambiguous elemental composition assignments, e.g., 1754 peaks (rms error = 175 ppb) corresponding to a paraffin series (C12–C49, double-bond equivalents, DBE = 0) and higher DBE series corresponding to cycloparaffins containing one to eight rings. Isoabundance-contoured plots of DBE versus carbon number highlight steranes (DBE = 4) of carbon number C27–C30 and hopanes of C29–C35 (DBE = 5), with sterane-to-hopane ratio in good agreement with field ionization (FI) mass spectrometry analysis, but performed at atmospheric pressure. The overall speciation of nonpolar, aliphatic hydrocarbon base oil species offers a promising diagnostic probe to characterize crude oil and its products.

An upper elemental compositional boundary for fossil hydrocarbons has previously been established as double-bond equivalents (i.e., DBE = rings plus double bonds) not exceeding 90% of the number of carbons. For heteroatom-containing fossil compounds, the 90% rule still applies if each N atom is counted as a C atom. The 90% rule eliminates more than 10% of the possible elemental compositions at a given mass for fossil database molecules. However, some synthetic compounds can fall outside the upper boundary defined for naturally occurring compounds. Their inclusion defines an “absolute” upper boundary as DBE (rings plus double bonds to carbon) equal to carbon number plus one, and applies to all organic compounds including fullerenes and other molecules containing no hydrogen. Finally, the DBE definition can fail for molecules with particular atomic valences. Therefore, we also present a generalized DBE definition that includes atomic valence to enable calculation of the correct total number of rings, double bonds, and triple bonds for heteroatom-containing compounds.

Pyrolysis of solid biomass, in this case pine pellets and peanut hulls, generates a hydrocarbon-rich liquid product (bio-oil) consisting of oily and aqueous phases. Here, each phase is characterized by negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to yield unique elemental compositions for thousands of compounds. Bio-oils are dominated by Ox species: few oxygens per molecule for the oily phase and many more oxygens per molecules for the aqueous phase. Thus, the increased oxygen content per molecule accounts for its water solubility. Peanut hull bio-oil is much more compositionally complex and contains more nitrogen-containing compounds than pine pellet bio-oil. Bulk C, H, N, O, and S measurements confirm the increased levels of nitrogen-containing species identified in the peanut hull pyrolysis oil by FT-ICR MS. The ability of FT-ICR MS to identify and assign unique elemental compositions to compositionally complex bio-oils based on ultrahigh mass resolution and mass accuracy is demonstrated.

An absorption-mode Fourier transform ion cyclotron resonance (FT-ICR) mass spectrum exhibits two types of baseline distortion: a slow periodic oscillation even where no signal peaks are present, and additional distortion near signal peaks and proportional to signal magnitude. These distortions interfere with automated peak-picking, unless the baseline systematic variation is much less than baseline rms random noise. We previously showed that the slow oscillation is removed by low-pass filtering. Here, we present a fast, robust, and automated algorithm that flattens the absorption-mode spectral baseline, even in the vicinity of signal peaks. The method begins by defining baseline data values, followed by linear interpolation to generate baseline data values between the defined values, then boxcar smoothing to generate a final baseline spectrum, and final subtraction of that baseline from the original spectrum to yield a baseline-flattened absorption-mode spectrum. We apply the algorithm to a crude oil spectrum (with 8000 peaks) and to a ribonuclease A protein spectrum (with multiply-charged ion isotopic distributions). We identify many more peaks (crude oil) without loss of mass accuracy, and obtain more accurate isotopic distributions (RNase A).Graphical abstractHighlights► Phasing an FT-ICR mass spectrum achieves up to 2× higher resolution, but introduces baseline “roll”. ► Here, we provide an algorithm that eliminates baseline roll. ► Peak-picking, mass resolution, and quantitation are all improved.

We report the use of unimolecular dissociation by infrared radiation for gaseous multiphoton energy transfer to determine relative activation energy (Ea,laser) for dissociation of peptide sequence ions. The sequence ions of interest are mass-isolated; the entire ion cloud is then irradiated with a continuous wave CO2 laser, and the first order rate constant, kd, is determined for each of a series of laser powers. Provided these conditions are met, a plot of the natural logarithm of kd versus the natural logarithm of laser power yields a straight line, whose slope provides a measure of Ea,laser. This method reproduces the Ea values from blackbody radiative dissociation (BIRD) for the comparatively large, singly and doubly protonated bradykinin ions (nominally y9 and y92+). The comparatively small sequence ion systems produce Ea,laser values that are systematic underestimates of theoretical barriers calculated with density functional theory (DFT). However, the relative Ea,laser values are in qualitative agreement with the mobile proton model and available theory. Additionally, novel protonated cyclic-dipeptide (diketopiperazine) fragmentation reactions are analyzed with DFT. FT-ICR MS provides access to sequence ions generated by electron capture dissociation, infrared multiphoton dissociation, and collisional activation methods (i.e., bn, ym, cn, zm• ions).

Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.

The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and 1F5, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and the results were compared to previous mapping by immunological and mutational analyses. Antibody 2B5 defines a conformational epitope, and 1F5 defines a linear epitope. Intact murine IgG antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen–monoclonal antibody (Ag–mAb) complexes. mAb-complexed and uncomplexed (free) rAna o 2 were then subjected to HDX. HDX instrumentation and automation were optimized to achieve high sequence coverage by protease XIII digestion. The regions protected from H/D exchange upon antibody binding overlap and thus confirm the previously identified epitope-bearing segments: the first extension of HDX monitored by mass spectrometry to a full-length antigen–antibody complex in solution.

Ion cyclotron resonance frequency, f, is conventionally converted to ion mass-to-charge ratio, m/z (mass “calibration”) by fitting experimental data spanning the entire detected m/z range to the relation, m/z = A/f + B/f2, to yield rms mass error as low as ∼200 ppb for ∼10 000 resolved components of a petroleum crude oil. Analysis of residual error versus m/z and peak abundance reveals that systematic errors limit mass accuracy and thus the confidence in elemental composition assignments. Here, we present a calibration procedure in which the spectrum is divided into dozens of adjoining segments, and a separate calibration is applied to each, thereby eliminating systematic error with respect to m/z. Further, incorporation of a third term in the calibration equation that is proportional to the magnitude of each detected peak minimizes systematic error with respect to ion abundance. Finally, absorption-mode data analysis increases mass measurement accuracy only after minimization of systematic errors. We are able to increase the number of assigned peaks by as much as 25%, while reducing the rms mass error by as much as 3-fold, for significantly improved confidence in elemental composition assignment.

We present a novel nonresonant laser-based matrix-free atmospheric pressure ionization technique, atmospheric pressure laser-induced acoustic desorption chemical ionization (AP/LIAD-CI). The technique decouples analyte desorption from subsequent ionization by reagent ions generated from a corona discharge initiated in ambient air or in the presence of vaporized toluene as a CI dopant at room temperature. Analyte desorption is initiated by a shock wave induced in a titanium foil coated with electrosprayed sample, irradiated from the rear side by high-energy laser pulses. The technique enables facile and independent optimization of the analyte desorption, ionization, and sampling events, for coupling to any mass analyzer with an AP interface. Moreover, the generated analyte ions are efficiently thermalized by collisions with atmospheric gases, thereby reducing fragmentation. We have coupled AP/LIAD-CI to ultrahigh-resolution FT-ICR MS to generate predominantly [M + H]+ or M+• ions to resolve and identify thousands of elemental compositions from organic mixtures as complex as petroleum crude oil distillates. Finally, we have optimized the AP/LIAD CI process and investigated ionization mechanisms by systematic variation of placement of the sample, placement of the corona discharge needle, discharge current, gas flow rate, and inclusion of toluene as a dopant.

Complex natural organic mixtures such as petroleum require ultrahigh mass spectral resolution to separate and identify thousands of elemental compositions. Here, we incorporate a custom-built, voltage-compensated ICR cell for Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), based on a prior design by Tolmachev to produce optimal mass resolution. The compensated ICR cell installed in a custom-built 9.4 T FTICR mass spectrometer consists of seven cylindrical segments with axial proportions designed to generate a dc trapping potential that approaches an ideal three-dimensional axial quadrupolar potential. However, the empirically optimized compensation voltages do not correspond to the most quadrupolar trapping field. The compensation electrodes minimize variation in the reduced cyclotron frequency by balancing imperfections in the magnetic and electric field. The optimized voltages applied to compensation electrodes preserve ion cloud coherence for longer transient duration by approximately a factor of 2, enabling separation and identification of isobaric species (compounds with the same nominal mass but different exact mass) common in petroleum, such as C3 vs SH4 (separated by 3.4 mDa) and SH313C vs 12C4 (separated by 1.1 mDa). The improved performance of the ICR cell provides more symmetric peak shape and better mass measurement accuracy. A positive ion atmospheric pressure photoionization (APPI) petroleum spectrum yields more than 26 000 assigned peaks, Fourier-limited resolving power of 800 000 at m/z 500 (6.6 s transient duration), and 124 part per billion root mean square (rms) error. The tunability of the compensation electrodes is critical for optimal performance.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ∼420 000 at m/z 2 593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum.

Valence parity provides a way to distinguish between N-terminal and C-terminal electron capture dissociation/electron transfer dissociation (ECD/ETD) product ions based on their number of hydrogen plus nitrogen atoms determined by accurate mass measurement and forms a basis for de novo peptide sequencing. The effect of mass accuracy (0.1–1 ppm error) on c′/z• overlap and unique elemental composition overlap is evaluated for a database of c′/z• product ions each based on all possible amino acid combinations and four subset databases containing the same c′ ions but with z• ions determined by in silico digestion with trypsin, Glu-C, Lys-C, or chymotrypsin. High mass accuracy reduces both c′/z• overlap and unique elemental composition overlap. Of the four proteases, trypsin offers slightly better discrimination between N- and C-terminal ECD/ETD peptides. Interestingly, unique elemental composition overlap curves for c′/c′ and z•/z• peptide ions exhibit discontinuities at certain nominal masses for 0.1–1.0 ppm mass error. Also, as noted in the companion article (Polfer et al. Anal. Chem.2011, DOI: 10.1021/ac201624t), the number of ECD/ETD product ion amino acid compositions as a function of nominal mass increases exponentially with mass but with a superimposed modulation due to higher prevalence of certain elemental compositions.

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO2-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO2 approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC–MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.

We report the first application of online LC-MS (liquid chromatography–mass spectrometry) characterization of algae polar lipids by nanoscale high-performance liquid chromatography followed by electrospray ionization and mass analysis with a linear ion trap (LTQ) coupled with 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ultrahigh FT-ICR mass resolution provides highly accurate mass measurement and resolves monoisotopic peaks from interfering components for unique determination of lipid elemental compositions. We establish the polar lipid profile of fatty acids, glycolipids, phospholipids, and betaine lipids for a green algae, Nannochloropsis oculata, which is highly prized for its oils suitable for biodiesel production. Lipid headgroup and fatty acid identification is based on accurate mass measured by the FT-ICR MS and collision-induced dissociation (CID) MS/MS in the LTQ. Unequivocal lipid composition is further confirmed from isotopic fine structure at baseline resolution—achievable only with ultrahigh resolution FT-ICR MS.

Here, we present the Predator data station, a control system for FT-ICR mass spectrometers that champions speed and experimental flexibility while simultaneously providing stability, ease of use, and the ability to integrate more advanced hardware as it becomes available. The Predator is the first FT-ICR MS data station comprised solely of fast PCI, PXI, and yet faster PXI Express-based commercial data acquisition hardware. Increased data transfer speed is required because recorded transient data count increases linearly at higher magnetic field (higher measured frequency) with extended transient duration for FT-ICR MS instruments. The application of new cell designs with additional compensation voltages, experimental techniques to increase resolution, and experimental techniques that minimize/reject variations in ion abundance exemplify the scope of recent Predator data station implementations. When the above techniques are applied simultaneously, the results give rise to sub-30 ppb rms mass error for 5250 assigned peaks in a petroleum FT-ICR mass spectrum.The Predator data station is designed for facile implementation with any FT-ICR MS instrument. The Predator hardware provides 17 analog voltage outputs and 18 digital TTL outputs synchronized to a single timing source. SWIFT, chirp, and single frequency excitation waveforms are generated by a 100 MSample/s arbitrary waveform generator with a minimum 32 MB of onboard memory and the potential of terabytes of virtual memory via first in-first out (FIFO) buffering. Transient detection is facilitated by a 2-channel, 100 MSample/s digitizer with a minimum of 32 MB of onboard memory per channel. FIFO buffering implementation allows TB transient collection as well. Commercial hardware, royalty-free software solutions, and commercially produced custom printed circuit boards (PCB) for the cell controller ensure open availability. The present data complement numerous extant publications: the Predator data station has been the sole data station for the National High Magnetic Field Laboratory (NHMFL) 9.4 T FT-ICR MS instrument since July 2004, and several additional Predator data stations are in operation elsewhere.Graphical abstractHighlights► We describe hardware and software to control FT-ICR MS experiments. ► Our data station controls excitation and detection of a time-domain ICR signal. ► We achieve mass resolving power of 20,000 for ion selection.

Natural lipid profiling can improve our current understanding of disease mechanism in a systems biology approach combining genomics, proteomics, and phenotypic changes. However, lipid profiling is complicated by the >10,000 combinations of polar head group, hydrocarbon chain length and degree of unsaturation/hydroxylation, and glycan composition and branching pattern. Here, we show how LC separation coupled with high resolution Fourier transform ion cyclotron resonance mass analysis can quickly narrow down the possible phospholipid and glycosphingolipid compositions. That approach necessitates resolution of mass differences as small as 1.8 mDa [12C213C1N1 (51.0064 Da) vs. H3O3 (51.0082 Da)] in phospholipids and 1.6 mDa [13C2S1H2 (59.9944 Da) vs. N2O2 (59.9960 Da)] in glycosphingolipids. For novel/unknown lipid species, high mass accuracy based Kendrick mass defect analysis enables quick grouping of related lipid species for subsequent tandem MS structural characterization. For sulfur-containing lipid species, high mass resolution can reveal isotopic fine structure to verify assignment.Graphical abstractResearch highlights▶ First resolution of the smallest mass differences in glycosphingolipids (1.6 mDa) and phospholipids (1.8 mDa). ▶ Ultrahigh mass accuracy FT-ICR MS enables quick assignment of phospholipids and glycosphingolipids from an accurate mass based lipid library. ▶ Kendrick mass defect analysis is vital for identification and characterization of previously unknown species or species not included in such a library. ▶ Resolution of isotopic fine structure reveals the presence (and number) of specific atoms in biological glycosphingolipids (e.g., sulfur).

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry relies upon linearity between the ion cyclotron excitation and the observed response. However, nonlinearities result from non-ideal applied electric and magnetic fields and Coulombic interactions. Here, we report nonlinear response at low excitation electric field magnitude due to Coulombic shielding. The measured ICR signal magnitude exhibits an excitation voltage threshold that increases monotonically with the number of shielding ions (i.e., unexcited ions). If shielding ions are not present, ICR signal magnitude versus excitation voltage is linear (e.g., for quadrupole-isolated ions of nearly a single m/z). Finally, we show that shielding results in a reduced cyclotron radius at low excitation voltage, resulting in an increased rate of transient decay; thereby exacerbating response nonlinearity and excitation threshold for long data acquisition period.Graphical abstractResearch highlights▶ A trapped ion packet can shield the ions so as to prevent rf resonant excitation in FT-ICR MS. ▶ In FT-ICR MS, higher resolution ion isolation requires a lower number of trapped ions. ▶ Coulombic shielding results in nonlinear FT-ICR response at low excitation electric field magnitude.

At sufficiently high mass accuracy, it is possible to distinguish phosphorylated from unmodified peptides by mass measurement alone. We examine the feasibility of that idea, tested against a library of all possible in silico tryptic digest peptides from the human proteome database. The overlaps between in silico tryptic digest phosphopeptides generated from known phosphorylated proteins (1–12 sites) and all possible unmodified human peptides are considered for assumed mass error ranges of ±10, ±50, ±100, ±1000, and ±10,000 ppb. We find that for mass error ±50 ppb, 95% of all phosphorylated human tryptic peptides can be distinguished from nonmodified peptides by accurate mass alone throughout the entire nominal mass range. We discuss the prospect of on-line LC MS/MS to identify phosphopeptide precursor ions in MS1 for selected dissociation in MS2 to identify the peptide and site(s) of phosphorylation.Graphical abstractHighlights► Peptide phosphorylation can be detected by mass alone. ► For mass error ±50 ppb, 95% of all phosphorylated human tryptic peptides can be distinguished from nonmodified peptides. ► Phosphopeptide precursor ions in MS1 can be selected for dissociation in MS2.

Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides unparalleled mass measurement accuracy and resolving power. However, propagation of the technique into new analytical fields requires continued advances in instrument speed and sensitivity. Here, we describe a substantial redesign of our custom-built 9.4 tesla FTICR mass spectrometer that improves sensitivity, acquisition speed, and provides an optimized platform for future instrumentation development. The instrument was designed around custom vacuum chambers for improved ion optical alignment, minimized distance from the external ion trap to magnetic field center, and high conductance for effective differential pumping. The length of the transfer optics is 30% shorter than the prior system, for reduced time-of-flight mass discrimination and increased ion transmission and trapping efficiency at the ICR cell. The ICR cell, electrical vacuum feedthroughs, and cabling have been improved to reduce the detection circuit capacitance (and improve detection sensitivity) 2-fold. The design simplifies access to the ICR cell, and the modular vacuum flange accommodates new ICR cell technology, including linearized excitation, high surface area detection, and tunable electrostatic trapping potential.

Radiofrequency (rf) multipole ion guides are widely used to transfer ions through the strong magnetic field gradient between source and analyzer regions of external source Fourier transform ion cyclotron resonance mass spectrometers. Although ion transfer as determined solely by the electric field in a multipole ion guide has been thoroughly studied, transfer influenced by immersion in a strong magnetic field gradient has not been as well characterized. Recent work has indicated that the added magnetic field can have profound effects on ion transfer, ultimately resulting in loss of ions initially contained within the multipole. Those losses result from radial ejection of ions due to transient cyclotron resonance that occurs when ions traverse a region in which the magnetic field results in an effective cyclotron frequency equal to the multipole rf drive frequency divided by the multipole order (multipole order is equal to one-half the number of poles). In this work, we describe the analytical basis for ion resonance in a rf multipole ion guide with superposed static magnetic field and compare with results of numerical trajectory simulations.

Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein−protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species (12Cc1Hh14Nn16Oo32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.

Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent-exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.

We combine liquid chromatography, electrospray ionization, and Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS) to determine the sugar composition, linkage pattern, and attachment sites of N-linked glycans. N-linked glycans were enzymatically released from glycoproteins with peptide N-glycosidase F, followed by purification with graphitized carbon cartridge solid-phase extraction and separation over a TSK-Gel Amide80 column under hydrophilic interaction chromatography (HILIC) conditions. Unique glycopeptide compositions were determined from experimentally measured masses for different combinations of glycans and glycopeptides. The method was validated by identifying four peptides glycosylated so as to yield 12 glycopeptides unique in glycan composition for the standard glycoprotein, bovine alpha-2-HS-glycoprotein. We then assigned a total of 137 unique glycopeptide compositions from 18 glycoproteins from fetal bovine serum, and the glycan structures for most of the assigned glycopeptides were heterogeneous. Highly accurate FT-ICR mass measurement is essential for reliable identification.

It has been known for 35 years that phase correction of FTICR data can in principle produce an absorption-mode spectrum with mass resolving power as much as a factor of 2 higher than conventional magnitude-mode display, an improvement otherwise requiring a (much more expensive) increase in magnetic field strength. However, temporally dispersed excitation followed by time-delayed detection results in steep quadratic variation of signal phase with frequency. Here, we present a robust, rapid, automated method to enable accurate broadband phase correction for all peaks in the mass spectrum. Low-pass digital filtering effectively eliminates the accompanying baseline roll. Experimental FTICR absorption-mode mass spectra exhibit at least 40% higher resolving power (and thus an increased number of resolved peaks) as well as higher mass accuracy relative to magnitude mode spectra, for more complete and more reliable elemental composition assignments for mixtures as complex as petroleum.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allows detailed characterization of complex petroleum samples at the level of elemental composition assignment. Ultrahigh-resolution (450 000−650 000 at m/z 500) enables identification of isobaric species that differ in mass by 3 mDa or less, and high mass accuracy (mass error of better than 300 ppb), combined with Kendrick mass sorting, allows for unambiguous molecular formula assignment to each of more than 10 000−20 000 peaks in each mass spectrum. Thus, it is possible to identify, sort, and monitor thousands of elemental compositions simultaneously, as a function of the boiling point. Here, the detailed FT-ICR MS characterization of an Athabasca bitumen heavy vacuum gas oil (HVGO) distillation series exposes the progression of heteroatom class, type (double bond equivalents (DBE), number of rings plus double bonds to carbon), and carbon number for tens of thousands of crude oil species, as a function of the boiling point. Specifically, we analyze a distillation series of Athabasca bitumen HVGO with cut temperatures from the initial boiling point (IBP) to 538 °C (in eight cuts) by atmospheric pressure photoionization (APPI), as well as positive and negative electrospray ionization (ESI) FT-ICR MS, to determine the distributions of nonpolar and polar species, as a function of the HVGO boiling point. Compositional distributions reveal definitive heteroatom class, type, and carbon number trends among distillation cuts, and provide the first detailed compositional evidence in support of the Boduszynski model that describes the progression of petroleum composition and structure as a function of the boiling point. Quantitation of the aromaticity and carbon number profiles of both polar and nonpolar species in all distillate cuts further affirms the validity of the Boduszynski model for the HVGO distillate range, and provides evidence for cycloalkane linkages, in addition to polyaromatic cores.

Heavy petroleum fractions are structurally and compositionally complex mixtures that defy characterization by many traditional analytical techniques. Here, we present the detailed characterization of a Middle Eastern heavy crude oil distillation series, in further support of the Boduszynski model, which proposes that petroleum is a continuum with regard to composition, molecular weight, aromaticity, and heteroatom content as a function of the boiling point. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolving power and mass accuracy and thereby allows for elemental assignment for each of the tens of thousands of peaks in a single crude oil sample. Part 1 of our five-part series established the validity of the Boduszynski model for the heavy vacuum gas oil (HVGO) distillation series. Here, we extend our analysis to fractions from a Middle Eastern heavy crude with cut temperatures including and beyond the middle distillate range. Collectively, the detailed compositional results for all heteroatom classes strongly support the continuity model. Interestingly, extrapolation of distillable compositional space to a high carbon number (up to 1 MDa) cannot account for the bulk properties of nondistillable (asphaltenic) species. Thus, either the continuity model does not accurately describe nondistillable petroleum materials (they are discontinuous in compositional space) or they are not high-molecular-weight (>2000 Da) materials.

Mass analysis of proteolytic fragment peptides following hydrogen/deuterium exchange offers a general measure of solvent accessibility/hydrogen bonding (and thus conformation) of solution-phase proteins and their complexes. The primary problem in such mass analyses is reliable and rapid assignment of mass spectral peaks to the correct charge state and degree of deuteration of each fragment peptide, in the presence of substantial overlap between isotopic distributions of target peptides, autolysis products, and other interferant species. Here, we show that at sufficiently high mass resolving power (m/Δm50% ≥ 100,000), it becomes possible to resolve enough of those overlaps so that automated data reduction becomes possible, based on the actual elemental composition of each peptide without the need to deconvolve isotopic distributions. We demonstrate automated, rapid, reliable assignment of peptide masses from H/D exchange experiments, based on electrospray ionization FT-ICR mass spectra from H/D exchange of solution-phase myoglobin. Combined with previously demonstrated automated data acquisition for such experiments, the present data reduction algorithm enhances automation (and thus expands generality and applicability) for high-resolution mass spectrometry-based analysis of H/D exchange of solution-phase proteins.

Atmospheric pressure photoionization (APPI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has significantly contributed to the molecular speciation of petroleum. However, a typical APPI source operates at 50 μL/min flow rate and thus causes a considerable mass load to the mass spectrometer. The recently introduced microchip APPI (μAPPI) operates at much lower flow rates (0.05−10 μL/min) providing decreased mass load and therefore decreased contamination in analysis of petroleum by FT-ICR MS. In spite of the 25 times lower flow rate, the signal response with μAPPI was only 40% lower than with a conventional APPI source. It was also shown that μAPPI provides very efficient vaporization of higher molecular weight components in petroleum analysis.

The GM2 activator protein (GM2AP) is an 18 kDa nonenzymatic accessory protein involved in the degradation of neuronal gangliosides. Genetic mutations of GM2AP can disrupt ganglioside catabolism and lead to deadly lysosomal storage disorders. Crystallography of wild-type GM2AP reveals 4 disulfide bonds and multiple conformations of a flexible loop region that is thought to be involved in lipid binding. To extend the crystallography results, a cysteine construct (L126C) was expressed and modified with 4-maleimide TEMPO for electron paramagnetic resonance (EPR) studies. However, because a ninth cysteine has been added by site-directed mutagenesis and the protein was expressed in E. coli in the form of inclusion bodies, the protein could misfold during expression. To verify correct protein folding and labeling, a sequential multiple-protease digestion, nano-liquid chromatograph (LC) electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry assay was developed. High-magnetic field and robust automatic gain control results in subppm mass accuracy for location of the spin-labeled cysteine and verification of proper connectivity of the four disulfide bonds. The sequential multiple protease digestion strategy and ultrahigh mass accuracy provided by FTICR MS allow for rapid and unequivocal assignment of relevant peptides and provide a simple pipeline for analyzing other GM2AP constructs.

Vanadyl porphyrins are detected and characterized by their double-bond equivalents (DBE = number of rings plus double bonds) and carbon number in an unfractionated (raw) asphaltene and unaltered South American crude oil. Atmospheric pressure photoionization (APPI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides the high mass-resolving power (450 000−650 000 at m/z 500) and accurate mass (<300 ppb) to unambiguously assign elemental compositions to each of more than 10 000 peaks in each mass spectrum. Kendrick mass sorting revealed unusually high mass errors for peaks assigned to high DBE O2 species as well as a suspicious bimodal distribution in plots of DBE versus carbon number for all O2 species. Inclusion of vanadium in the chemical formula assignment resolved the bimodal distribution into lower DBE O2 species and vanadyl porphyrins, with a subsequent decrease in mass assignment errors to the same level as those for the thousands of other identified species. Vanadyl porphyrins are detected as both M+ • and [M + H]+ molecular and quasimolecular ions. Trends in the relative abundance of specific DBE values reveal the structural diversity of the vanadyl porphyrins in the asphaltene and heavy crude oil. To our knowledge, the current results are the first to directly identify and catalog the structural diversity of vanadyl porphyrins directly in raw (unfractionated) asphaltene and unaltered heavy crude oil.

In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein–protein or protein–ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages roughly 30% because of the long exposure to H2O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.A 2-min sham digestion indicates no backbone back exchange during digestion for fully exchanged LHRH. Rapid chromatography with a ProZap™ C18 column reduces back exchange by about one third compared to a Jupiter™ C5 column.Figure optionsDownload full-size imageDownload high-quality image (150 K)Download as PowerPoint slide

Calcium and sodium naphthenates are solid deposits and emulsions formed by the interaction of naphthenic acids with divalent (Ca2+, Mg2+) or monovalent (Na+, K+) ions in produced waters. Calcium naphthenate formation, an interfacial phenomenon, is thought to depend largely on tetraprotic naphthenic acids known as “ARN” acids (∼C80) in the crude oil, whereas sodium naphthenates originate from lower molecular weight (C15 to C35) monoprotic naphthenic acids. Here we present detailed chemical heteroatom class composition analyses of calcium and sodium naphthenates from the field based on high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In all cases, calcium naphthenate deposits consist predominately of tetraprotic acids with a C80 hydrocarbon skeleton whereas sodium naphthenate emulsions consist mainly of specific monoprotic saturated carboxylic acids. Furthermore, low molecular weight tetraprotic (ARN) acids with C60−77 hydrocarbon skeletons were identified in the calcium naphthenate deposit. The high resolution and mass accuracy of FT-ICR MS provide detailed acidic speciation for the analyzed deposits and emulsions.

We examine suspected molecular transformations of thermally treated Athabasca bitumen heavy vacuum gas oil (HVGO) by ultrahigh-resolution negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Liquid products from HVGOs treated under an inert N2 atmosphere at temperatures of 300, 325, 350, and 400 °C were each characterized by class (heteroatom content), type (double-bond equivalents = number of rings plus double bonds to carbon), and carbon number distribution. In addition, the inert N2 sweep gas of the autoclave was collected, condensed, and analyzed. The total acid number (TAN) of the HVGO liquid products decreases with an increasing treatment temperature (from 4.13 at 300 °C to 1.46 at 400 °C), indicative of potential carboxylic acid decomposition. The highly abundant O2 class contains species with DBE = 3; however, no compositional changes occur with increased treatment temperature. A bimodal DBE distribution is observed for the S1O2 class, suggesting two possible stable core structures. Only low relative abundance classes show slight changes with thermal treatment. Condensed nitrogen sweep gas obtained at 350 and 400 °C contains highly abundant O2 species with DBE of 3 and but at lower carbon number. Similarly, the condenser product S1O2 classes display the same bimodal DBE distributions as the HVGO liquid products but with lower carbon number (∼18−27 for condenser versus ∼25−35 for the liquid products). The similarity of the O2 speciation in the HVGO liquid products after thermal treatment combined with the detailed analysis of the condenser products suggests that the gross decrease in total acid number (TAN) at higher temperature is due to global (class, DBE, and carbon number indiscriminant) decomposition of the naphthenic acids as well as a small contribution from the loss of the lower boiling, lower carbon number acids by simple distillation.

We describe a novel SIMION model, based on the principle of reciprocity, to simulate ion–image charge detection and demonstrate utility for Fourier transform ion cyclotron resonance mass spectrometry. The model accommodates arbitrary electrode geometry, magnetic field inhomogeneity, ion-neutral collisions, and swept or single-frequency excitation, but does not account for ion–ion or ion–image charge forces. Accurate frequency-domain spectra reflect actual features of FT-ICR mass spectra including odd and even harmonic multiples of the ion cyclotron frequency, sidebands at odd and even multiples of the trapping frequency, and asymmetric sidebands at even multiples of the magnetron frequency. The model is general and could be applied to other mass analyzers including the quadrupole ion trap, time-of-flight, and orbitrap.A SIMION model accurately calculates ion image charge detection in FT-ICR mass spectrometry that reveals nonlinear spectral features including ICR harmonics, trapping sidebands, and asymmetric magnetron sidebands.

Analysis of petroleum samples at the molecular level by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) typically requires a prolonged accumulation of ions and/or summing up a large number of scans. Here, a chip-based micro-ESI system (Advion NanoMate, Ithaca, NY) has been successfully automated in combination with FT-ICR MS analysis of petroleum samples. A foil-sealed 96-well glass plate prevents solvent evaporation, with no visible loss of sample after 20 h of continuous operation. Mass spectra obtained from the same sample but taken from different wells after various time delays were very similar. Data from replicate samples in different wells could be combined to enhance mass spectral signal-to-noise ratio and dynamic range. Furthermore, the automated data acquisition eliminates sample carryover, and produces heteroatom class distribution, double-bond equivalents (DBE), and carbon number very similar to those from the conventional (manual) micro-ESI experiments.

Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry offers a rapid method to study protein conformations and protein−protein interactions. Pepsin is usually used to digest proteins in HDX and is known for lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FTICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein−protein interactions.

We describe the design and current performance of a 14.5 T hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer. Ion masses are routinely determined at 4-fold better mass accuracy and 2-fold higher resolving power than similar 7 T systems at the same scan rate. The combination of high magnetic field and strict control of the number of trapped ions results in external calibration broadband mass accuracy typically less than 300 ppb rms, and a resolving power of 200 000 (m/Δm50% at m/z 400) is achieved at greater than 1 mass spectrum per second. Novel ion storage optics and methodology increase the maximum number of ions that can be delivered to the FTICR cell, thereby improving dynamic range for tandem mass spectrometry and complex mixture applications.

We describe automation of liquid injection field desorption/ionization (LIFDI) for reproducible sample application, improved spectral quality, and high-throughput analyses. A commercial autosampler provides reproducible and unattended sample application. A custom-built field desorption (FD) controller allows data station or front panel control of source parameters including high-voltage limit/ramp rate, emitter heating current limit/ramp rate, and feedback control of emitter heating current based on ion current measurement. Automated LIFDI facilitates ensemble averaging of hundreds of Fourier transform ion cyclotron resonance mass spectra for increased dynamic range, mass accuracy, and S/N ratio relative to single-application FD experiments, as shown here for a South American crude oil. This configuration can be adapted to any mass spectrometer with an LIFDI probe.

Because acids in petroleum materials are known to corrode processing equipment, highly acidic oils are sold at a discount [on the basis of their total acid number (TAN)]. Here, we identify the acidic species in raw Canadian bitumen (Athabasca oil sands) and its distilled heavy vacuum gas oil (HVGO) as well as acid-only and acid-free fractions isolated by use of an ion-exchange resin (acid−IER) and negative-ion electrospray ionization Fourier transform ion cyclotron resonance (ESI FT−ICR MS) mass spectrometry. The ultrahigh mass resolving power ( m/Δ m 50% > 400 000) and high mass accuracy (better than 500 ppb) of FT−ICR MS, along with Kendrick mass sorting, enable the assignment of a unique elemental composition to each peak in the mass spectrum. Acidic species are characterized by class (N n O o S s heteroatom content), type [number of rings plus double bonds to carbon or double-bond equivalent (DBE)], and carbon number distribution. We conclude that the analytical capability of FT−ICR MS and the selectivity of the ESI process eliminate the need for acid fractionation to characterize naphthenic acids in bitumen. However, because the acid-free fraction (not retained on the acid−IER) contains S x O y heteroatomic classes not observed in the parent bitumen, acid−IER fractionation does help to identify such low-abundance species. Further, we observe that a subset of the acids identified in the parent bitumen distill into the HVGO fraction. Variations in the carbon number and aromaticity of the classes are discussed in detail.

We have analyzed eight heavy vacuum gas oil (HVGO) distillation fractions, initial boiling point (IBP)−343, 343−375, 375−400, 400−425, 425−450, 450−475, 475−500, and 500−525 °C, of an Athabasca bitumen by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Acidic, basic, and nonpolar components were detected by negative-ion and positive-ion electrospray ionization (ESI) and automated liquid injection field desorption ionization (LIFDI) positive-ion FT-ICR MS. Ultrahigh mass resolving power (m/Δm50% ≈ 350 000) and high mass accuracy (<500 ppb) facilitate the assignment of a unique elemental composition to each peak in the mass spectrum. Thus, each distillate was characterized by mass, heteroatom class, type (number of rings and double bonds), and carbon number distribution to correlate compositional changes with increased boiling point. Negative-ion ESI FT-ICR MS identifies high relative abundance nonaromatic O2 species that span the entire distillation range. All ionization methods reveal an increase in double-bond equivalents (DBE, the number of rings plus double bonds) and carbon number with increased distillation temperature. In addition, some structural information can be inferred from increases in DBE value with increased distillation temperature. Summed data for individual distillation cuts yield class specific isoabundance contours similar to that for the feed HVGO, suggesting that class-specific carbon number and DBE distributions for individual distillation cuts could be estimated from the high-resolution feed HVGO mass spectrum.

We examine oil-specific asphaltene inhibitor chemistry of two chemically distinct asphaltene inhibitors. Laboratory and field tests show oil-specific asphaltene inhibitor performance for two geographically distinct crude oils. The crude oils and their corresponding asphaltenes were characterized by total acid number (TAN), elemental analysis, Fourier transform infrared spectroscopy (FTIR), and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT−ICR MS). ESI FT−ICR MS reveals differences in the relative abundance of heteroatom-containing compound classes in the two crude oils and the two asphaltenes. We identify acidic and/or basic species that may be responsible for the observed differences in inhibitor chemistry. Asphaltene inhibitor specificity can be explained by acid−base-type interactions between the inhibitor and polar species in the crude oils or asphaltene fractions. ESI FT−ICR MS provides the first evidence for inhibitor effectiveness related to heteroatom content derived from detailed polar chemical composition.

Each different molecular elemental composition—e.g., CcHhNnOoSs—has a different exact mass. With sufficiently high mass resolving power (m/Δm50% ≈ 400,000, in which m is molecular mass and Δm50% is the mass spectral peak width at half-maximum peak height) and mass accuracy (<300 ppb) up to ≈800 Da, now routinely available from high-field (≥9.4 T) Fourier transform ion cyclotron resonance mass spectrometry, it is possible to resolve and identify uniquely and simultaneously each of the thousands of elemental compositions from the most complex natural organic mixtures, including petroleum crude oil. It is thus possible to separate and sort petroleum components according to their heteroatom class (NnOoSs), double bond equivalents (DBE = number of rings plus double bonds involving carbon, because each ring or double bond results in a loss of two hydrogen atoms), and carbon number. “Petroleomics” is the characterization of petroleum at the molecular level. From sufficiently complete characterization of the organic composition of petroleum and its products, it should be possible to correlate (and ultimately predict) their properties and behavior. Examples include molecular mass distribution, distillation profile, characterization of specific fractions without prior extraction or wet chemical separation from the original bulk material, biodegradation, maturity, water solubility (and oil:water emulsion behavior), deposits in oil wells and refineries, efficiency and specificity of catalytic hydroprocessing, “heavy ends” (asphaltenes) analysis, corrosion, etc.

To further clarify the role of dopant solvent in proton transfer in atmospheric pressure photoionization (APPI), we employ ultrahigh-resolution FT-ICR mass analysis to identify M+•, [M+H]+, [M-H]−, and [M+D]+ species in toluene or perdeuterotoluene for an equimolar mixture of five pyrrolic and pyridinic nitrogen heterocyclic model compounds, as well as for a complex organic mixture (Canadian Athabasca bitumen middle distillate). In the petroleum sample, the protons in the [M+H]+ species originate primarily from other components of the mixture itself, rather than from the toluene dopant. In contrast to electrospray ionization, in which basic (e.g., pyridinic) species protonate to form [M+H]+ positive ions and acidic (e.g., pyrrolic) species deprotonate to form [M-H]− negative ions, APPI generates ions from both basic and acidic species in a single positive-ion mass spectrum. Ultrahigh-resolution mass analysis (in this work, m/Δm50%=500,000, in which Δm50% is the mass spectral peak full width at half-maximum peak height) is needed to distinguish various close mass doublets: 13C versus 12CH (4.5 mDa), 13CH versus 12CD (2.9 mDa), and H2 versus D (1.5 mDa).

We determine the elemental compositions of aromatic nitrogen model compounds as well as a petroleum sample by atmospheric pressure photoionization (APPI) and electrospray Ionization (ESI) with a 9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. From the double-bond equivalents calculated for the nitrogen-containing ions from a petroleum sample, we can infer the aromatic core structure (pyridinic versus pyrrolic nitrogen heterocycle) based on the presence of M+· (odd-electron) versus [M+H]+ (even-electron) ions. Specifically, nitrogen speciation can be determined from either a single positive-ion APPI spectrum or two ESI (positive- and negative-ion) spectra. APPI operates at comparatively higher temperature than ESI and also produces radical cations that may fragment before detection. However, APPI fragmentation of aromatics can be eliminated by judicious choice of instrumental parameters.

Electron capture dissociation (ECD) efficiency in a 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer varies periodically with the time interval between ion and electron injection. The observed modulation frequency correlates to within 1% with ion magnetron frequency, most probably due to misalignment between the ion beam and the electron beam. The optimum ECD conditions are obtained by correctly phasing electron injection with the ion magnetron motion. Displacement of the trapped ion cloud by variation of the ICR trap radial electric field decreases ECD efficiency modulation amplitude. Experiments directly suggest that only ions interacting with electrons at the moment of electron injection participate in ECD reactions.

The diminishing clean oil reserve is driving the search for new or improved ways to reduce the level of NSO-containing species found in high abundance in heavy crude oils. Hydrotreatment is the currently preferred technique to remove those polar species. Unfortunately, nitrogen-containing compounds cause coke formation on the surface of the hydrotreatment catalyst, leading to partial or complete deactivation. Here, positive- and negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) identify those nitrogen compounds that resist hydrotreatment. ESI preferentially ionizes polar (e.g., heteroatom-containing) species: basic molecules are detected as positive ions and acidic/neutral molecules as negative ions. FT-ICR MS resolves thousands of species in a single mass spectrum, allowing for unambiguous determination of elemental composition, CcHhNnOoSs, for identification of compound “class” (numbers of N, O, S heteroatoms, “type” (rings plus double bonds), and carbon number (revealing the extent of alkylation). We find that hydrotreatment-resistant compounds typically contain a single nitrogen atom, both pyridinic benzalogs and pyrollic benzalogs. Compounds with more than one heteroatom, such as Ox, NxOy, NxSy and Nx, are partially removed. Compound classes with lower double bond equivalents or fewer CH2 groups are preferentially removed. Species that contain OxSy are fully removed by hydrotreatment.

Kalata peptides are isolated from an African medicinal plant, Oldenlandia affinis, an aqueous decoction of which can be ingested to accelerate uterine contraction during childbirth. The closely packed disulfide core of kalata peptides confers unusual stability against thermal, chemical, and enzymatic degradation. The molecular arrangement may hamper NMR-assisted disulfide connectivity assignment. We have combined NMR with high-resolution mass spectrometry (MS) and MS/MS of native and chemically derivatized kalata B2 to determine its amino acid sequence and disulfide connectivity. Infrared multiphoton dissociation establishes the disulfide bond linkages in kalata B2 as I–IV, II–V and III–VI.

Electron capture dissociation (ECD) efficiency has typically been lower than for other dissociation techniques. Here we characterize experimental factors that limit ECD and seek to improve its efficiency. Efficiency of precursor to product ion conversion was measured for a range of peptide (∼15% efficiency) and protein (∼33% efficiency) ions of differing sizes and charge states. Conversion of precursor ions to products depends on electron irradiation period and maximizes at ∼5–30 ms. The optimal irradiation period scales inversely with charge state. We demonstrate that reflection of electrons through the ICR cell is more efficient and robust than a single pass, because electrons can cool to the optimal energy for capture, which allows for a wide range of initial electron energy. Further, efficient ECD with reflected electrons requires only a short (∼500 μs) irradiation period followed by an appropriate delay for cooling and interaction. Reflection of the electron beam results in electrons trapped in or near the ICR cell and thus requires a brief (∼50 μs) purge for successful mass spectral acquisition. Further electron irradiation of refractory precursor ions did not result in further dissociation. Possibly the ion cloud and electron beam are misaligned radially, or the electron beam diameter may be smaller than that of the ion cloud such that remaining precursor ions do not overlap with the electron beam. Several ion manipulation techniques and use of a large, movable dispenser cathode reduce the possibility that misalignment of the ion and electron beams limits ECD efficiency.

A suite of six genetically related oils that had experienced varying degrees of subsurface, anaerobic biodegradation was analyzed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry. By use of electrospray ionization of whole oil samples, all neutral nitrogen compounds and acid NSO compounds, from ∼300 to 900 Da, are selectively characterized and assigned unambiguous molecular formulae. Several methods of data visualization reveal changes in the relative abundances of these compounds with increasing degradation.Evidence for selective biodegradation is observed in all compound classes. NSO compounds associated with long alkyl side chains are removed, regardless of the NSO core, under conditions associated with moderate (saturated biomarkers unaffected) to severe biodegradation. Some compound series, such as O1, O3, and SO3–9, are mineralized under conditions of mild biodegradation (n-alkanes, isoprenoids altered but still present). Changes in the Z-series (hydrogen deficiency) and alkyl distributions of the O2 species result from simultaneous microbial degradation and generation. Acyclic fatty acids decrease, whereas Z = −10 O2 species, which correspond to five-ring naphthenic (hopanoic) acids, increase in relative abundance during early stages of biodegradation. Monocyclic (Z = −2) O2 are enriched initially, and then decrease during advanced stages of biodegradation. O2 species corresponding to di-, tri-, and tetra-cyclic naphthenic acids (Z = −4, −6 and −8) are preferentially produced during these advanced stages. The ratio of acyclic to 2–4 ring cyclic O2-species provides a new parameter to define the degree of biodegradation.

We have applied electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to analyze the pyridine soluble fraction of a distillation resid and a further processed liquid product in a coal liquefaction process. The inherent high resolving power (m/Δm50%>300,000, in which Δm50% is full mass spectral peak width at half-maximum peak height) and mass accuracy (<1 ppm) of FT-ICR MS makes it possible to resolve and identify polar heteroatomic species. The resid contains more heteroatomic compounds and a higher molecular weight distribution whereas the liquid sample is lower in average mass and more saturated. The data confirms that the liquefaction process produces lower mass, hydrogenated liquid product whereas the resid (highly aromatic and of high heteroatom content) must be recycled to reduce its heteroatom content and increase its degree of saturation.

Inspection of the electron capture dissociation (ECD) spectra of doubly-protonated peptides, Leu4-Sar-Leu3-Lys-OH, Leu4-Ala-Leu3-Lys-OH, Gly4-Sar-Gly3-Lys-NH2 and Gly3-Pro-Sar-Gly3-Lys-NH2, reveals extensive secondary fragmentation. In addition to w ions, entire, and in some cases multiple, cleavages of amino acid side chains from backbone fragments are observed. Extensive water loss from backbone fragments is observed for the glycine-rich peptides. For Leu4-Ala-Leu3-Lys, the preferred fragmentation channel is cleavage of the amide bond to produce b7 and b8 ions. ECD of Gly3-Pro-Sar-Gly3-Lys-NH2 results in amine bond (c/z) cleavage in the proline residue accompanied by CC (or secondary NC) cleavage in the proline side chain. That fragmentation channel has not been observed previously. The peptides were also subjected to “hot” electron capture dissociation (HECD) and the resulting spectra differed markedly from those obtained under standard ECD conditions. In contrast to HECD, secondary fragmentation observed under standard ECD conditions cannot be attributed to excess energy arising from the kinetic energy of the electrons prior to capture. The results suggest that the fragmentation channels available following electron capture depend somewhat on the individual peptide structure and have mechanistic implications.

We present the selective ionization, resolution and identification of acidic NSO compounds in three crude oils of different geochemical origins by negative ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Selective ionization by ESI affords direct detection of neutral nitrogen compounds and carboxylic acids in petroleum without pre-chromatographic isolation. Ultra-high resolution/mass accuracy allows detailed and positive identification of acidic NSO compounds in the crude oils. Observed compositional differences reflect known crude oil properties/histories. Collectively, ∼14,000 masses, spanning 18 different heteroatomic classes, are identified unequivocally, demonstrating the potential of ESI-FT-ICR MS for geochemical applications.

We have used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry to characterize amino acid side chain losses observed during electron capture dissociation (ECD) of ten 7- to 14-mer peptides. Side-chain cleavages were observed for arginine, histidine, asparagine or glutamine, methionine, and lysine residues. All peptides containing an arginine, histidine, asparagine or glutamine showed the losses associated with that residue. Methionine side-chain loss was observed for doubly-protonated bombesin. Lysine side-chain loss was observed for triply-protonated dynorphin A fragment 1–13 but not for the doubly-protonated ion. The proximity of arginine to a methoxy C-terminal group significantly enhances the extent of side-chain fragmentation. Fragment ions associated with side-chain losses were comparable in abundance to those resulting from backbone cleavage in all cases. In the ECD spectrum of one peptide, the major product was due to fragmentation within an arginine side chain. Our results suggest that cleavages within side chains should be taken into account in analysis of ECD mass spectral data. Losses from arginine, histidine, and asparigine/glutamine can be used to ascertain their presence, as in the analysis of unknown peptides, particularly those with non-linear structures.

Externally generated ions are accumulated in a linear octopole ion trap before injection into our 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer. Such instrumental configuration has previously been shown to provide improved sensitivity, scan rate, and duty cycle relative to accumulated trapping in the ICR cell. However, inefficient ion ejection from the octopole currently limits both detection limit and scan rate. SIMION 7.0 analysis predicts that a dc axial electric field inside the linear octopole ion trap expedites and synchronizes the efficient extraction of the octopole-accumulated ions. Further SIMION analysis optimizes the ion ejection properties of each of three electrode configurations designed to produce a near-linear axial potential gradient. More efficient extraction and transfer of accumulated ions spanning a wide m/z range promises to reduce detection limit and increase front-end sampling rate (e.g., to increase front-end resolution for separation techniques coupled with FT-ICR mass analysis). Addition of the axial field improves experimental signal-to-noise ratio by more than an order of magnitude.

In this paper, we report negative ion microelectrospray Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry of C60 samples containing ∼1% 3He@C60 or 4He@C60. Resolving 3He@C60− and 4He@C60− from C60 containing 3 or 4 13C instead of 12C atoms is technically challenging, because the target species are present in low relative abundance and are very close in mass. Nevertheless, we achieve baseline resolution of 3He@C60− from 13C312C57− and 4He@C60− from 13C412C56− in single-scan mass spectra obtained in broadband mode without preisolation of the ions of interest. The results constitute the first direct mass spectrometric observation of endohedral helium in a fullerene sample at this (low) level of incorporation. The results also demonstrate the feasibility of determining the extent of He incorporation from the FT-ICR mass spectral peak heights. The present measurements are in agreement with those obtained by the pyrolysis method 1; 2 ;  3. Although limited in sensitivity, the mass spectral method is faster and easier than pyrolysis.

This communication demonstrates that gentle infrared laser heating can remove unwanted buffer adducts from a gas-phase protein complex without dissociating the complex itself. Specifically, noncovalent complexes of the oligopeptide-binding protein, OppA, bound to either (Ala)3 or LysTrpLys were electrosprayed from aqueous buffer solution into a 9.4 tesla Fourier transform ion cyclotron resonance mass spectrometer. In addition to the intact complexes, several additional buffer adduct species were produced under the conditions of the experiment. Irradiation of the trapped ion population with a continuous-wave infrared CO2 laser at relatively low power (2.5 W) for 1 s dissociated the buffer adducts but retained the intact protein:peptide complexes. Adduct-free complex(es) were then readily identified, and signal-to-noise ratio also increased by an order of magnitude because the same number of protein ions are distributed over fewer species. Higher IR power (5 W for 1 s) dissociated the adduct-free complex(es) without internal fragmentation. The present in-trap clean-up technique may prove especially useful for identifying and screening the combinatorial library ligands most strongly bound to a receptor in the gas phase.

By comparing electrospray ionization Fourier-transform ion cyclotron resonance (FT-ICR) mass spectra and collision-induced dissociation (CID) FT-ICR mass spectra of a phospholipid (851 Da) extracted from natural abundance and 99% 13C bacterial growth media, we are able to reduce its number of possible elemental compositions (based on ±10 ppm externally calibrated mass accuracy and biologically relevant compositional constraints) from 394 to 1. The basic idea is simply that the mass of a molecule containing N carbon atoms increases by N Da when 12C is replaced by 13C. Once the number of carbons is known, the number of possible combinations of other atoms in the molecule is greatly reduced. We demonstrate the method for a stored-waveform inverse Fourier transform-isolated phospholipid from an extract of membrane lipids from Rhodococcus rhodochrous hydrocarbon-degrading bacteria grown on either natural abundance or 99% 13C-enriched mixtures of n-hexadecane and n-octadecane. We project that this method raises the upper mass limit for unique determination of elemental composition from accurate mass measurement by a factor of at least 3, thereby extending “chemical formula” determination to identification and sequencing of larger synthetic and bio-polymers: phospholipids, oligopeptides of more than three to four amino acids, DNA or RNA of more than two nucleotides, oligosaccharides of more than three sugars, etc. The method can also be extended to determination of the number of other atoms for which heavy isotopes are available (e.g., 15N, 34S, 18O, etc.).

Unambiguous determination of metal atom oxidation state in an intact metalloprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic abundance mass distributions for one or more holoprotein charge states. The iron atom oxidation state is determined unequivocally as Fe(III) for each of four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobin, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution-phase oxidation state is conserved following electrospray to produce gas-phase ions). However, the same Fe(III) oxidation state in all four heme proteins is observed after prior reduction by sodium dithionite to produce Fe(II) heme proteins in solution: thus proving that oxygen was present during the electrospray process. Those results bear directly on the issue of similarity (or lack thereof) of solution-phase and gas-phase protein conformations. Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holoproteins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe(II)heme from the covalently bound heme in cytochrome c.

In a perfect three-dimensional axial quadrupolar electrostatic potential field, Ledford et al. showed that the frequency-to-mass calibration relation m/z = AL/v + BL/v2is valid for ions of any mass-to-charge ratio, m/z < (m/z)critical = eB02a2/4Vtrapα, in which v is the “reduced” (observed) ion cyclotron frequency, e is the electronic (elementary) charge, z is the number of elementary charges per ion, B0 is magnetic field induction, a is a characteristic trap dimension, vtrap is the potential applied to each trap endcap, α is a constant determined by the trap geometrical configuration, and AL and BL are constants that are determined by fitting experimental ion cyclotron resonance (ICR) frequencies for ions of at least two known masses in a Fourier transform ICR (FT-ICR) mass spectrum. In the further limit that m/z ≪ (m/z)critical, Francl et al. obtained a different frequency-to-mass relation m/z = AF/(BF+ v). Here, we rederive both frequency-to-mass relations to derive a simple conversion between ALand BL, versus AFand BF(e.g. for comparing calibrated FT-ICR mass spectral data from different vendors). For accurate mass measurement, the conversion introduces a small error (a few parts per billion) that can usually be neglected. More important, by applying both calibration equations to the same experimental time-domain data, we find that mass accuracy resulting from the two calibration functions (or their interconversion) is indistinguishable, because Ledford et al.’s validity criterion, m/z < 0.001 (m/z)critical, is generally satisfied for modern high-field instruments with optimized cell geometry. Interestingly, a small difference may result when different forms of the same calibration function are employed, presumably due to different roundoff errors in the calculation.

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.

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 ± 86 Å2 for apo-cNTnC to 3108 ± 71 Å2 for Ca2+-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca2+-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.

A different symmetry is required to optimize each of the three most common Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) electric potentials in a Penning (ICR) ion trap: one-dimensional dipolar ac for excitation (or detection), two-dimensional azimuthal quadrupolar ac excitation for ion axialization, and three-dimensional axial quadrupolar dc potential for ion axial confinement (trapping). Since no single trap shape simultaneously optimizes all three potentials, many trap configurations have been proposed to optimize the tradeoffs between the three requirements for a particular experiment. A more general approach is to divide each electrode into small segments and then apply the appropriate potential to each segment. Here, we extend segmentation to its logical extreme, by constructing a “matrix-shimmed” trap consisting of a cubic trap, with each side divided into a 5 × 5 grid of electrodes for a total of 150 electrodes. Theoretically, only 48 independent voltages need be applied to these 150 electrodes to generate all three desired electric potential fields simultaneously. In practice, it is more convenient to employ 63 independent voltages due to construction constraints. Resistive networks generate the potentials required for optimal quadrupolar trapping and quadrupolar excitation. To avoid resistive loss of excitation amplitude and detected signal, dipolar excitation/detection voltages are generated with a capacitive network. Theoretical Simion 6.0 simulations confirm the achievement of near-ideal potentials of all three types simultaneously. From a proof-of-principle working model, several experimental benefits are demonstrated, and proposed future improvements are discussed.

Gas-phase hydrogen/deuterium exchange of D2O with [M + H]+ ions of angiotensin II, angiotensin I, [Sar1]-angiotensin II, bradykinin, des-Arg1-bradykinin, des-Arg9-bradykinin, luteinizing hormone releasing hormone (LH-RH), and substance P has been examined by Fourier transform ion cyclotron resonance mass spectrometry at 9.4 tesla. Because the FTICR dynamic range increases quadratically with magnetic field, parent ions from a mixture of several peptides may be confined simultaneously for long periods at high pressure (e.g., 1 h at 1 × 10−5 torr) without quadrupolar axialization (and its attendant ion heating), for faster data acquisition and better controlled comparisons between different peptides. A high magnetic field also facilitates stored waveform inverse Fourier transform (SWIFT) isolation of monoisotopic [M + H]+ parent ions, so that deuterium incorporation patterns may be determined directly without the need for isotopic distribution deconvolution. Finally, a higher magnetic field provides for a greatly extending trapping period, for measurement of much slower rates. Angiotensin I, angiotensin II, and [Sar1]-angiotensin II are found to undergo a rapid exchange. Angiotensin II and [Sar1]-angiotensin II exhibit multiple deuterium uptake distributions, corresponding to multiple gas-phase conformations. In contrast, substance P exchanges slowly and LH-RH displays no observable exchange. Comparison of the relative H/D exchange rates for bradykinin and its des-Arg-derivatives supports the hypothesis that bradykinin adopts a folded gas-phase conformation that unfolds upon removal of either terminal arginine residue.

Laser-induced ion fluorescence of laser-desorbed Ba+ ions provides a measure of the relative number of ions near the center of the Penning trap of a Fourier transform ion cyclotron resonance mass spectrometer. Here, we report the detection of Penning-trapped ions by ion fluorescence, subject to radially outward ion cloud expansion (because of ion–neutral collisions), radially inward ion cloud compression (because of quadrupolar axialization), and the effects of buffer gas pressure and electrostatic trapping potential on those processes. At high pressure and high trapping voltage, radial ejection is far more rapid than axial ejection; quadrupolar axialization increases the number of ions near the center of the trap as well as the length of time that ions may be trapped; higher pressure results in faster magnetron radial expansion; and the choice of azimuthal quadrupolar excitation waveform significantly affects the efficacy of axialization. Based on these results, we suggest that directly detected laser-induced ion fluorescence provides a general new tool for mapping the ion distribution and its time evolution in response to various excitatory and damping effects.

To further clarify the role of dopant solvent in proton transfer in atmospheric pressure photoionization (APPI), we employ ultrahigh-resolution FT-ICR mass analysis to identify M+•, [M + H]+, [M − H]−, and [M + D]+ species in toluene or perdeuterotoluene for an equimolar mixture of five pyrrolic and pyridinic nitrogen heterocyclic model compounds, as well as for a complex organic mixture (Canadian Athabasca bitumen middle distillate). In the petroleum sample, the protons in the [M + H]+ species originate primarily from other components of the mixture itself, rather than from the toluene dopant. In contrast to electrospray ionization, in which basic (e.g., pyridinic) species protonate to form [M + H]+ positive ions and acidic (e.g., pyrrolic) species deprotonate to form [M − H]− negative ions, APPI generates ions from both basic and acidic species in a single positive-ion mass spectrum. Ultrahigh-resolution mass analysis (in this work, m/Δm50% = 500,000, in which Δm50% is the mass spectral peak full width at half-maximum peak height) is needed to distinguish various close mass doublets: 13C versus 12CH (4.5 mDa), 13CH versus 12CD (2.9 mDa), and H2 versus D (1.5 mDa).

We determine the elemental compositions of aromatic nitrogen model compounds as well as a petroleum sample by atmospheric pressure photoionization (APPI) and electrospray Ionization (ESI) with a 9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. From the double-bond equivalents calculated for the nitrogen-containing ions from a petroleum sample, we can infer the aromatic core structure (pyridinic versus pyrrolic nitrogen heterocycle) based on the presence of M+ ·  (odd-electron) versus [M+H]+ (even-electron) ions. Specifically, nitrogen speciation can be determined from either a single positive-ion APPI spectrum or two ESI (positive- and negative-ion) spectra. APPI operates at comparatively higher temperature than ESI and also produces radical cations that may fragment before detection. However, APPI fragmentation of aromatics can be eliminated by judicious choice of instrumental parameters.

Analysis of petroleum samples at the molecular level by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) typically requires a prolonged accumulation of ions and/or summing up a large number of scans. Here, a chip-based micro-ESI system (Advion NanoMate, Ithaca, NY) has been successfully automated in combination with FT-ICR MS analysis of petroleum samples. A foil-sealed 96-well glass plate prevents solvent evaporation, with no visible loss of sample after 20 h of continuous operation. Mass spectra obtained from the same sample but taken from different wells after various time delays were very similar. Data from replicate samples in different wells could be combined to enhance mass spectral signal-to-noise ratio and dynamic range. Furthermore, the automated data acquisition eliminates sample carryover, and produces heteroatom class distribution, double-bond equivalents (DBE), and carbon number very similar to those from the conventional (manual) micro-ESI experiments.Photos of the unassembled multiple-sample holder, prior to installation in a NanoMate robot.Download high-res image (200KB)Download full-size image

Mass analysis of proteolytic fragment peptides following hydrogen/deuterium exchange offers a general measure of solvent accessibility/hydrogen bonding (and thus conformation) of solution-phase proteins and their complexes. The primary problem in such mass analyses is reliable and rapid assignment of mass spectral peaks to the correct charge state and degree of deuteration of each fragment peptide, in the presence of substantial overlap between isotopic distributions of target peptides, autolysis products, and other interferant species. Here, we show that at sufficiently high mass resolving power (m/Δm50% ≥ 100,000), it becomes possible to resolve enough of those overlaps so that automated data reduction becomes possible, based on the actual elemental composition of each peptide without the need to deconvolve isotopic distributions. We demonstrate automated, rapid, reliable assignment of peptide masses from H/D exchange experiments, based on electrospray ionization FT-ICR mass spectra from H/D exchange of solution-phase myoglobin. Combined with previously demonstrated automated data acquisition for such experiments, the present data reduction algorithm enhances automation (and thus expands generality and applicability) for high-resolution mass spectrometry-based analysis of H/D exchange of solution-phase proteins.New automated software enables correct assignment of isotopic distribution fine structure in H/D exchange experiments monitored by ultrahigh-resolution mass spectrometry.Download high-res image (162KB)Download full-size image

It is known that the ion collision cross section (CCS) may be calculated from the linewidth of a Fourier transform ion cyclotron resonance (FT-ICR) mass spectral peak at elevated pressure (e.g., ∼10−6 Torr). However, the high mass resolution of FT-ICR is sacrificed in those experiments due to high buffer gas pressure. In this study, we describe a linewidth correction method to eliminate the windowing-induced peak broadening effect. Together with the energetic ion–neutral collision model previously developed by our group, this method enables the extraction of CCSs of biomolecules from high-resolution FT-ICR mass spectral linewidths, obtained at a typical operating buffer gas pressure of modern FT-ICR instruments (∼10−10 Torr). CCS values of peptides including MRFA, angiotensin I, and bradykinin measured by the proposed method agree well with ion mobility measurements, and the unfolding of protein ions (ubiquitin) at higher charge states is also observed.