Fourier transform ion cyclotron resonance: state of the art.

This short review summarizes recent and projected advances in Fourier transform ion cyclotron resonance mass spectrometry instrumentation and applications, ranging from petroleomics to proteomics. More details are available from the cited primary literature and topical reviews.

Overview of Target Enrichment Strategies.

Target enrichment is commonly used in next generation sequencing (NGS) workflows to eliminate genomic DNA regions that are not of interest for a particular experiment. By only targeting specific regions such as exons, one can obtain greater depth of DNA sequencing coverage for regions of interest or increase the sampling numbers of individuals, thereby saving both time and cost. This overview of target enrichment strategies provides a high-level review of distinct approaches to capture specific sequences: (a) hybridization-based strategies, (b) transposon-mediated fragmentation (tagmentation), (c) molecular inversion probes (MIPs), and (d) singleplex and multiplex polymerase chain reaction (PCR) target enrichment. Strategies for assay design and performance criteria are also discussed. Other platforms currently in development are also briefly described.

Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe(III)myoglobin, Fe(III)cytochrome c, Fe(III)cytochrome b5, and Fe(III)cytochrome b5 L47R.

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 ion 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.

Two-dimensional coulomb-induced frequency modulation in Fourier transform ion cyclotron resonance: A mechanism for line broadening at high mass and for large ion populations.

Fourier transform ion cyclotron resonance (FTICR) spectra generated for large ion populations exhibit frequency shifts and line broadening, apparently due to Coulomb forces between ions. Although previous two-dimensional (2D) models of Coulomb effects in FTICR accounted for frequency shifts, they did not account for spectral line broadening. In this article, a 2D model is proposed that predicts line broadening due to Coulomb-induced frequency modulation. The model considers the case of two different-mass ions orbiting at their respective cyclotron frequencies around a common guiding center. A mutual modulation of the cyclotron frequency occurs at the difference frequency between ions. If the modulation period is much shorter than the FTICR observation time, then sidebands spaced at intervals approximately equal to the modulation frequency are predicted. However, if the modulation period is similar in duration to the FTICR observation period, the sidebands can no longer be resolved, which results in spectral line broadening. This latter case is a necessary consequence for isotopic peaks in the high mass region around m/z 2000, where deterioration in FTICR performance has been observed. Computer simulations are used to confirm the mass dependence and to demonstrate other features of the model, including a strong dependence of the modulation on ion number. In support of the model, experimental FTICR spectra for large populations of methylnaphthalene ions at m/z 141 and 142 exhibit constant frequency sidebands corresponding to multiples of the difference frequency for the two ions extending from nominal values of m/z 136 to 147.

High-resolution electrospray ionization Fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus Stearothermophilus.

Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.

Simplified application of quadrupolar excitation in Fourier transform ion cyclotron resonance mass spectrometry.

A new method for application of quadrupolar excitation to the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is presented. Quadrupolar excitation is conventionally applied to the two pairs of opposed electrodes that normally perform the excitation and detection functions in the FTICR experiment. Symmetry arguments and numerically calculated isopotential contours within the trapped ion cell lead to the conclusion that quadrupolar excitation can be applied to a single pair of opposed side electrodes. Examples of effective quadrupolar axialization via this method include a sevenfold signal-to-noise enhancement derived from 50 remeasurements of a single population of trapped bovine insulin ions and the selective isolation of a single charge state of horse heart myoglobin after an initial measurement that revealed the presence of 14 charge states.

Ion kinetic energy modulation for improved ion trapping in electrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

A new technique for manipulating the kinetic energy distribution of electrospray ions that arrive at a Fourier transform ion cyclotron resonance trapped-ion cell is presented. Narrow kinetic energy distributions can complicate the selection of appropriate trapping conditions for electrospray ions and introduce charge discrimination in resulting mass spectra. Modulation of the applied skimmer potential controllably broadens the kinetic energy distribution, which improves the reproducibility of acquired spectra and eliminates charge discrimination. Mass spectra of horse heart cytochrome c are presented to demonstrate the utility of the technique. For example, applied static skimmer potentials of 12 and 9 V yield charge state distributions ranging from [M+19H](+19) to [M+12H](+12) and [M+15H](+15) to [M+7H](+7), respectively. A 12 ± 2 V, 100-Hz modulation of the skimmer potential yields an electrospray spectrum with charge states that range from [M+19H](+19) to [M+7H](+7), which is more representative of the source distribution.

Stable isotope incorporation triples the upper mass limit for determination of elemental composition by accurate mass measurement.

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.).

Competitive binding to the oligopeptide binding protein, OppA: in-trap cleanup in an Fourier transform ion cyclotron resonance mass spectrometer.

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.

Application of micro-electrospray liquid chromatography techniques to FT-ICR MS to enable high-sensitivity biological analysis.

A microbore electrospray (ESI) injection system has been adapted to our 9.4-tesla ESI FT-ICR mass spectrometer, greatly enhancing the stability and sensitivity of the system. Spray was generated from micro-ESI needles made from sharply tapered, polished fused silica capillaries of 25 to 50 microns inner diameter. Micro-ESI permits low-level sample analysis by constant infusion at sub-microL/min flow rate over a wide range of solvent conditions in both positive- and negative-ion mode. The system is flexible and allows rapid conversion to allow routine LC/MS analysis on low-level mixtures presented in biological media. LC/MS analyses were accomplished by replacing micro-ESI needles with capillaries packed with reverse phase retention media to permit analyte concentration and purification prior to analysis (micro-ESI/LC). A unique nano-flow LC pumping system was developed, capable of producing a true unsplit solvent gradient at flow rates below 1 microL/min. The micro-ESI/LC FT-ICR system produces mass spectra from a mixture of three neuroactive peptides at a concentration of 500 amol/microL (5 fmol each total loaded) in biological salts with baseline separation, signal-to-noise ratio of > 10:1 and mass resolving power > 5000. These results represent a reduction in detection limit by a factor of approximately 2 x 10(6) over the best previously published LC/FT-ICR MS data.

Note: Optimized circuit for excitation and detection with one pair of electrodes for improved Fourier transform ion cyclotron resonance mass spectrometry.

A conventional Fourier transform-Ion Cyclotron Resonance (ICR) detection cell is azimuthally divided into four equal sections. One pair of opposed electrodes is used for ion cyclotron excitation, and the other pair for ion image charge detection. In this work, we demonstrate that an appropriate electrical circuit facilitates excitation and detection on one pair of opposed electrodes. The new scheme can be used to minimize the number of electrically independent ICR cell electrodes and/or improve the electrode geometry for simultaneously increased ICR signal magnitude and optimal post-excitation radius, which results in higher signal-to-noise ratio and decreased space-charge effects.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The basic principles and recent advances in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are reviewed. A brief history of electrospray ionization is provided, along with a complete technical description of the technique, electrospray ionization variations, and advantages. Next, the fundamental principles of Fourier transform ion cyclotron resonance mass spectrometry are covered, including ion cyclotron motion, ion cyclotron resonance excitation, and image current detection. Instrumentation and methods used to couple these techniques are then described. Topics include ion source configuration, ion transport through a strong magnetic field gradient, and ion trapping methods. The article concludes with selected applications that highlight the strengths of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Baseline mass resolution of peptide isobars: a record for molecular mass resolution.

Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, approximately 904 Da, has been achieved by microelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at a mass resolving power of approximately 3 300 000. The elemental compositions of these molecules differ by N40 vs. S2H8 (0.000 45 Da), which is less than one electron's mass (0.000 55 Da)! This result establishes a new record for the smallest resolved mass difference between any two molecules. This achievement is made possible by a combination of high magnetic field (9.4 T), large-diameter (4-in.) Penning trap, and low ion density. The implications for proteomics based on accurate mass measurements are discussed briefly.

Fourier transform ion cyclotron resonance mass spectrometry: a primer.

This review offers an introduction to the principles and generic applications of FT-ICR mass spectrometry, directed to readers with no prior experience with the technique. We are able to explain the fundamental FT-ICR phenomena from a simplified theoretical treatment of ion behavior in idealized magnetic and electric fields. The effects of trapping voltage, trap size and shape, and other nonidealities are manifested mainly as perturbations that preserve the idealized ion behavior modified by appropriate numerical correction factors. Topics include: effect of ion mass, charge, magnetic field, and trapping voltage on ion cyclotron frequency; excitation and detection of ICR signals; mass calibration; mass resolving power and mass accuracy; upper mass limit(s); dynamic range; detection limit, strategies for mass and energy selection for MSn; ion axialization, cooling, and remeasurement; and means for guiding externally formed ions into the ion trap. The relation of FT-ICR MS to other types of Fourier transform spectroscopy and to the Paul (quadrupole) ion trap is described. The article concludes with selected applications, an appendix listing accurate fundamental constants needed for ultrahigh-precision analysis, and an annotated list of selected reviews and primary source publications that describe in further detail various FT-ICR MS techniques and applications.

Posttranslational heterocyclization of cysteine and serine residues in the antibiotic microcin B17: distributivity and directionality.

To produce the antibiotic Microcin B17, four Cys and four Ser residues are converted into four thiazoles and four oxazoles by the three subunit Microcin B17 synthetase. High-resolution mass spectrometry (MS) was used to monitor the kinetics of posttranslational heterocyclic ring formation (-20 Da per ring) and demonstrated the accumulation of all intermediates, from one to seven rings, indicating distributive processing. All of the intermediates could be converted by the enzyme to the eight ring product. Enzymatic chemoselectivity (Cys vs Ser cyclization rates) was assessed using iodoacetamido-salicylate to alkylate unreacted cysteines (+193 Da) in the 8 kDa biosynthetic intermediates; three of the first four rings formed were thiazoles, and by the five ring stage, all four of the cysteines had been heterocyclized while three of the original four serines remained uncyclized. Finally, tandem MS using a 9.4 T Fourier transform instrument with electrospray ionization was used to elaborate the major processing pathway: the first two rings formed are at the most amino proximal sites (Cys(41) then Ser(40)) followed by the remaining three cysteines at positions 48, 51, and 55. The cyclization of serines at positions 56, 62, and 65 then follows, with Ser(62) and Ser(65) the last to heterocyclize and the first of these at a slower rate. Thus, despite free dissociation of intermediates after each of seven ring-forming catalytic cycles, there is an overall directionality of ring formation from N-terminal to C-terminal sites. This remarkable regioselectivity is determined more by the substrate than the enzyme, due to a combination of (1) initial high-affinity binding of the posttranslational catalyst to the N-terminal propeptide of substrate 88mer, and (2) a chemoselectivity for thiazole over oxazole formation. This mechanism is consistent with antibiotic biosynthesis in vivo, yielding microcin with six, seven, and eight rings, all with bioactivity.

Counting individual sulfur atoms in a protein by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry: experimental resolution of isotopic fine structure in proteins.

A typical molecular ion mass spectrum consists of a sum of signals from species of various possible isotopic compositions. Only the monoisotopic peak (e.g., all carbons are 12C; all nitrogens are 14N, etc.) has a unique elemental composition. Every other isotope peak at approximately integer multiples of approximately 1 Da higher in nominal mass represents a sum of contributions from isotope combinations differing by a few mDa (e.g., two 13C vs. two 15N vs. one 13C and one 15N vs. 34S, vs. 18O, etc., at approximately 2 Da higher in mass than the monoisotopic mass). At sufficiently high mass resolving power, each of these nominal-mass peaks resolves into its isotopic fine structure. Here, we report resolution of the isotopic fine structure of proteins up to 15.8 kDa (isotopic 13C,15N doubly depleted tumor suppressor protein, p16), made possible by electrospray ionization followed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass analysis at 9.4 tesla. Further, a resolving power of m/Deltam50% approximately 8,000,000 has been achieved on bovine ubiquitin (8.6 kDa). These results represent a 10-fold increase in the highest mass at which isotopic fine structure previously had been observed. Finally, because isotopic fine structure reveals elemental composition directly, it can be used to confirm or determine molecular formula. For p16, for example, we were able to determine (5.1 +/- 0.3) the correct number (five) of sulfur atoms solely from the abundance ratio of the resolved 34S peak to the monoisotopic peak.

High-sensitivity electron capture dissociation tandem FTICR mass spectrometry of microelectrosprayed peptides.

Electron capture dissociation (ECD) has previously been shown by other research groups to result in greater peptide sequence coverage than other ion dissociation techniques and to localize labile posttranslational modifications. Here, ECD has been achieved for 10-13-mer peptides microelectrosprayed from 10 nM (10 fmol/microL) solutions and for tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. Tandem Fourier transform ion cyclotron resonance (FTICR) mass spectra contain fragment ions corresponding to cleavages at all possible peptide backbone amine bonds, except on the N-terminal side of proline, for substance P and neurotensin. For luteinizing hormone-releasing hormone, all but two expected backbone amine bond cleavages are observed. The tandem FTICR mass spectra of the tryptic peptides contain fragment ions corresponding to cleavages at 6 of 12 (1545.7-Da peptide) and 8 of 21 (2944.5-Da peptide) expected backbone amine bonds. The present sensitivity is 200-2000 times higher than previously reported. These results show promise for ECD as a tool to produce sequence tags for identification of peptides in complex mixtures available only in limited amounts, as in proteomics.

Kendrick mass defect spectrum: a compact visual analysis for ultrahigh-resolution broadband mass spectra.

At currently achievable Fourier transform ion cyclotron resonance broadband mass spectrometry resolving power (m/deltam50% > 350,000 for 200 < m/z < 1,000), it would be necessary to spread out a conventional mass spectrum over approximately 200 m in order to provide visual resolution of the most closely resolved peaks. Fortunately, there are natural gaps in a typical mass spectrum, spaced 1 Da apart, because virtually no commonly encountered elemental compositions yield masses at those values. Thus, it is possible to break a broadband mass spectrum into 1-Da segments, rotate each segment by 90 degrees, scale each segment according to its mass defect (i.e., difference between exact and nominal mass), and then compress the spacing between the segments to yield a compact display. For hydrocarbon systems, conversion from IUPAC mass to "Kendrick" mass (i.e., multiplying each mass by 14.00000/14.01565) further simplifies the display by rectilinearizing the peak patterns. The resulting display preserves not only the "coarse" spacings (e.g., approximately 1 Da between odd and even masses, corresponding to either even vs odd number of nitrogens or 12C(c) vs 12C(c-1)13C1 elemental compositions of the same molecule; approximately 2-Da separations, corresponding to a double bond or ring; approximately 14 Da separations, corresponding to one CH2 group) but also the "fine structure" (i.e., different mass defects for different elemental compositions) across each 1-Da segment. The method is illustrated for experimental electrospray ionization FTICR ultrahigh-resolution mass spectra of a petroleum crude oil. Several thousand elemental compositions may be resolved visually in a single one-page two-dimensional display, and various compound families-class (NnOoSs), type (Z in C(c)H2(c+z)NnOoSs), and alkylation series-may be identified visually as well.

Fourier transform ion cyclotron resonance mass spectrometry in a 20 T resistive magnet.

We present Fourier transform ion cyclotron resonance (FTICR) mass spectra at a magnetic field of 20 T; more than twice the highest field previously used for FTICR. Our instrument is based on a resistive magnet installed at the National High Magnetic Field Laboratory. The magnet has a 50 mm diameter bore and spatial inhomogeneity of approximately 1000 ppm over a 1 cm diameter spherical volume. However, FTICR mass resolving power far in excess of magnet homogeneity is achieved routinely for ions produced by either electron ionization (EI) or matrix-assisted laser desorption/ionization (MALDI). As examples, we show a MALDI mass spectrum of [M + H] quasimolecular ions of the peptide, human luteinizing hormone-releasing hormone (monoisotopic molecular weight, 1181.6 Da) at mass resolving power, m/delta m > 10,000; and an EI mass spectrum of molecular ions of the platinum cluster compound, Pt4(PF3)8 (average molecular weight, 1484 Da at mass resolving power, m/delta m approximately 20,000. Much better FTICR MS performance is predicted for future NHMFL resistive magnets of higher spatial and temporal homogeneity.

Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.