Donald F. Smith

Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe–4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g_∥ = 2.035 and g_⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at g_iso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5′-deoxyadenosyl radical (5′-dAdo•). The time course of the 5′-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5′-dAdo• by Fe–C5′ bond homolysis, and the 5′-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5′-dAdo• intermediate trapped just prior to reaction with the substrate.

High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry provides the highest mass resolving power and mass measurement accuracy for detailed characterization of complex chemical mixtures. Here, we report the coupling of online liquid chromatography of complex mixtures with a 21 tesla FT-ICR mass spectrometer. The high magnetic field enables large ion populations to be analyzed for each spectrum for a high dynamic range, with 3.2 million mass resolving power at m/z 400 (6.2 s transient duration) or 1.6 million (3.1 s transient duration) while maintaining high mass accuracy for molecular formula assignment (root-mean-square assignment error < 0.150 ppm). Thousands of unique elemental compositions are assigned per mass spectrum, which can be grouped by the heteroatom class, double bond equivalents (the number of rings and double bonds to carbon), and carbon number. Figures of merit are discussed, as well as characterization of an Arabian heavy vacuum gas oil in terms of the ring number, compound class, double bond equivalents, and ion type. Consideration of elemental composition and retention order provides additional structural information.

Petroleum derived dissolved organic matter (DOM_HC) samples were successfully cationized with barium, revealing many [M–H + Ba]⁺ peaks in both dark and simulated sunlight treatments. The DOM_HC samples generated after light exposure exhibited a greater number of [M–H + Ba]⁺ peaks compared to the dark control. Multiple [M–H + Ba]⁺ peaks were investigated in the irradiated DOM_HC using low resolution MS/MS in order to confirm the presence of diagnostic fragment ions, m/z 139, 155 and 196 in each treatment. Due to the high complexity of the bariated DOM_HC mixture, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS/MS) was employed to obtain molecular level information for both irradiated and dark treatments. The irradiated DOM_HC treatments had more bariated oxygenated species over a wide range of H/C and O/C when compared to the dark controls. Doubly bariated species were also observed in DOM_HC, which provides evidence that photochemistry transforms DOM_HC to even more complex mixtures with multiple oxygenations per molecule. This study provides evidence that barium adduct mass spectrometry can be successfully applied to DOM_HC screening for the presence of COOHs, both in dark samples and solar irradiated samples. Furthermore, direct evidence and molecular composition of aqueous phase crude oil photoproducts is provided by this technique.

This work is the second installment of a study that probes the aggregation behavior of asphaltenes by gel permeation chromatography (GPC). In part 1, analysis of GPC aggregate fractions collected from the 2017 PetroPhase asphaltene sample by direct infusion revealed an inverse correlation between aggregate size and aromaticity. However, characterization of the largest aggregate fractions by direct infusion was hampered by solvent contaminant peaks and dynamic range limitations due to the extremely low ionization efficiencies of the larger, more aliphatic species that comprise those fractions. Here, we couple the GPC separation with online detection by positive atmospheric pressure photoionization ((+)APPI) 21 tesla Fourier transform ion cyclotron resonance mass spectrometry (21 T FT-ICR MS) to overcome those problems and reveal that the most abundant species that comprise the largest aggregate segment are indeed the most aliphatic. The ability to characterize difficult-to-analyze samples, like asphaltenes, is the first major advantage of online coupling. Another benefit is the increased chromatographic resolution afforded by online coupling, which enables a finer examination in the most aggregated region and reveals a local trend opposed to the global trend. The very first species to elute in the largest aggregates were more condensed polyaromatic compounds, and the larger, more aliphatic species elute shortly thereafter in much greater relative abundance.

Stored waveform inverse Fourier transform (SWIFT) is a versatile method to generate complex isolation/ejection waveforms for precursor isolation prior to tandem mass spectrometry experiments. Here, we report ultrahigh resolving power ion isolation by SWIFT on a 21 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Individual histone proteoforms are isolated (0.6 m/z isolation window) with near 100% efficiency using a 52 ms SWIFT isolation, followed by in-cell fragmentation by ultraviolet photodissociation (UVPD). Ion isolation resolving power of 175 000 (m/Δm) is demonstrated by isolation of individual peaks at a spacing of 0.0034 Da at m/z 597 from a complex mixture of Canadian bitumen. An individual m/z ion, which corresponds to a single elemental composition, from a complex mixture is isolated and fragmented by infrared multiphoton dissociation (IRMPD). Theoretical and experimental considerations that limit achievable ion isolation resolving power are discussed.

Detailed characterization of complex biological surfaces by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) requires instrumentation that is capable of high mass resolving power, mass accuracy, and dynamic range. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers the highest mass spectral performance for MALDI MSI experiments, and often reveals molecular features that are unresolved on lower performance instrumentation. Higher magnetic field strength improves all performance characteristics of FT-ICR; mass resolving power improves linearly, while mass accuracy and dynamic range improve quadratically with magnetic field strength. Here, MALDI MSI at 21T is demonstrated for the first time: mass resolving power in excess of 1,600,000 (at m/z 400), root-mean-square mass measurement accuracy below 100 ppb, and dynamic range per pixel over 500:1 were obtained from the direct analysis of biological tissue sections. Molecular features with m/z differences as small as 1.79 mDa were resolved and identified with high mass accuracy. These features allow for the separation and identification of lipids to the underlying structures of tissues. The unique molecular detail, accuracy, sensitivity, and dynamic range combined in a 21T MALDI FT-ICR MSI experiment enable researchers to visualize molecular structures in complex tissues that have remained hidden until now. The instrument described allows for future innovative, such as high-end studies to unravel the complexity of biological, geological, and engineered organic material surfaces with an unsurpassed detail.

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/Δm_50% > 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 O_o, S_sO_o, and NO_o 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 C_cH_hN_nO_oS_s mass differences at high mass (m/z > 600). Similar performance gains are realized in the analysis of dissolved organic matter, where doubly charged O_o species are resolved from singly charged SO_o 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.