Greg T. Blakney

Automated broadband phase correction of Fourier transform ion cyclotron resonance mass spectra.

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.

Epitope mapping of a 95 kDa antigen in complex with antibody by solution-phase amide backbone hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

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.

Fourier transform ion cyclotron resonance mass resolution and dynamic range limits calculated by computer modeling of ion cloud motion.

Particle-in-Cell (PIC) ion trajectory calculations provide the most realistic simulation of Fourier transform ion cyclotron resonance (FT-ICR) experiments by efficient and accurate calculation of the forces acting on each ion in an ensemble (cloud), including Coulomb interactions (space charge), the electric field of the ICR trap electrodes, image charges on the trap electrodes, the magnetic field, and collisions with neutral gas molecules. It has been shown recently that ion cloud collective behavior is required to generate an FT-ICR signal and that two main phenomena influence mass resolution and dynamic range. The first is formation of an ellipsoidal ion cloud (termed “condensation”) at a critical ion number (density), which facilitates signal generation in an FT-ICR cell of arbitrary geometry because the condensed cloud behaves as a quasi-ion. The second phenomenon is peak coalescence. Ion resonances that are closely spaced in m/z coalesce into one resonance if the ion number (density) exceeds a threshold that depends on magnetic field strength, ion cyclotron radius, ion masses and mass difference, and ion initial spatial distribution. These two phenomena decrease dynamic range by rapid cloud dephasing at small ion density and by cloud coalescence at high ion density. Here, we use PIC simulations to quantitate the dependence of coalescence on each critical parameter. Transitions between independent and coalesced motion were observed in a series of the experiments that systematically varied ion number, magnetic field strength, ion radius, ion m/z, ion m/z difference, and ion initial spatial distribution (the present simulations begin from elliptically-shaped ion clouds with constant ion density distribution). Our simulations show that mass resolution is constant at a given magnetic field strength with increasing ion number until a critical value (N) is reached. N dependence on magnetic field strength, cyclotron radius, ion mass, and difference between ion masses was determined for two ion ensembles of different m/z, equal abundance, and equal cyclotron radius. We find that N and dynamic range depend quadratically on magnetic field strength in the range 1–21 Tesla. Dependences on cyclotron radius and Δm/z are linear. N depends on m/z as (m/z)–2. Empirical expressions for mass resolution as a function of each of the experimental parameters are presented. Here, we provide the first exposition of the origin and extent of trade-off between FT-ICR MS dynamic range and mass resolution (defined not as line width, but as the separation between the most closely resolved masses).

Automated electrospray ionization FT-ICR mass spectrometry for petroleum analysis

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.

Petroleomics: a test bed for ultra-high- resolution Fourier transform ion cyclotron resonance mass spectrometry

Within a relative abundance dynamic range of ~10,000: 1, the worlds most compositionally complex organic mixture is petroleum crude oil. As such, it provides the most challenging target for mass spectral resolution and identification of molecules below m/z 2000. The mass “splits” in petroleum include most of those that also appear in proteomics, metabolomics and other complex organic mixture analysis. Therefore, petroleum provides an excellent test bed for optimizing mass spectrometer performance in general. The presence of multiple elemental compositions spanning less than 1 Da in mass facilitates mapping and correction of rf phase variation across a Fourier transform ion cyclotron resonance mass spectrum, as well as exposing otherwise inaccessible systematic mass deviations, for additional improvement in mass resolving power and mass accuracy by a factor of up to 5. Internal mass calibration, combined with systematic peak assignment for successive homologous series, enables automated elemental composition assignment of tens of thousands of peaks in a single mass spectrum.

Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry

Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.

Effect of magnetic field inhomogeneity on ion cyclotron motion coherence at high magnetic field

A three-dimensional code based on the particle-in-cell algorithm modified to account for the inhomogeneity of the magnetic field was applied to determine the effect of Z1, Z2, Z3, Z4, X, Y, ZX, ZY, XZ2 YZ2, XY and X2–Y2 components of an orthogonal magnetic field expansion on ion motion during detection in an FT-ICR cell. Simulations were performed for magnetic field strengths of 4.7, 7, 14.5 and 21 Tesla, including experimentally determined magnetic field spatial distributions for existing 4.7 T and 14.5 T magnets. The effect of magnetic field inhomogeneity on ion cloud stabilization (“ion condensation”) at high numbers of ions was investigated by direct simulations of individual ion trajectories. Z1, Z2, Z3 and Z4 components have the largest effect (especially Z1) on ion cloud stability. Higher magnetic field strength and lower m/z demand higher relative magnetic field homogeneity to maintain cloud coherence for a fixed time period. The dependence of mass resolving power upper limit on Z1 inhomogeneity is evaluated for different magnetic fields and m/z. The results serve to set the homogeneity requirements for various orthogonal magnetic field components (shims) for future FT-ICR magnet design.

21 Tesla FT-ICR Mass Spectrometer for Ultrahigh-Resolution Analysis of Complex Organic Mixtures

We describe complex organic mixture analysis by 21 tesla (T) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ultrahigh mass-resolving power (m/Δm50% > 2 700 000 at m/z 400) and mass accuracy (80 ppb rms) enable resolution and confident identification of tens of thousands of unique elemental compositions. We demonstrate 2.2-fold higher mass-resolving power, 2.6-fold better mass measurement accuracy, and 1.3-fold more assigned molecular formulas compared to our custom-built, state-of-the-art 9.4 T FT-ICR mass spectrometer for petroleum and dissolved organic matter (DOM) analyses. Analysis of a heavy petroleum distillate exemplifies the need for ultrahigh-performance mass spectrometry (49 040 assigned molecular formulas for 21 T versus 29 012 for 9.4 T) and extends the identification of previously unresolved Oo, SsOo, and NOo classes. Mass selective ion accumulation (20 Thompson isolation) of an asphalt volcano sample yields 462 resolved mass spectral peaks at m/z 677 and reveals previously unresolved CcHhNnOoSs mass differences at high mass (m/z > 600). Similar performance gains are realized in the analysis of dissolved organic matter, where doubly charged Oo species are resolved from singly charged SOo species, which requires a mass-resolving power greater than 1 400 000 (at m/z 600). This direct comparison reveals the continued need for higher mass-resolving power and better mass accuracy for comprehensive molecular characterization of the most complex organic mixtures.