Unequivocal Determination of Site-Specific Protein Disulfide Bond Reduction Potentials by Top-Down FTICR MS: Characterization of the N- and C‑Terminal Redox-Active Sites in Human Thioredoxin 1

We report the reliable determination of equilibrium protein disulfide bond reduction potentials (<i>E</i>°′) 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 <i>E. coli</i> thioredoxin, and we determined <i>E</i>°′ for its CGPC active-site disulfide as −280 mV in accord with literature values. <i>E</i>°′ 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 <i>S</i>-glutathionylation of Trx1 and localized that redox modification to Cys72; <i>E</i>°′ 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

Polar Lipid Composition of Biodiesel Algae Candidates <i>Nannochloropsis oculata</i> and <i>Haematococcus pluvialis</i> from Nano Liquid Chromatography Coupled with Negative Electrospray Ionization 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

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, <i>Nannochloropsis oculata</i> and <i>Haematococcus pluvialis</i>, 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. <i>H. pluvialis</i> exhibits more highly polyunsaturated lipids than does <i>N. oculata</i>

126 264 Assigned Chemical Formulas from an Atmospheric Pressure Photoionization 9.4 T Fourier Transform Positive Ion Cyclotron Resonance Mass Spectrum

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 <i>any</i> spectrum of any kind (e.g., UV–vis, infrared, microwave, magnetic resonance, etc.)

126 264 Assigned Chemical Formulas from an Atmospheric Pressure Photoionization 9.4 T Fourier Transform Positive Ion Cyclotron Resonance Mass Spectrum

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 <i>any</i> spectrum of any kind (e.g., UV–vis, infrared, microwave, magnetic resonance, etc.)

126 264 Assigned Chemical Formulas from an Atmospheric Pressure Photoionization 9.4 T Fourier Transform Positive Ion Cyclotron Resonance Mass Spectrum

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 <i>any</i> spectrum of any kind (e.g., UV–vis, infrared, microwave, magnetic resonance, etc.)

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism

Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in <i>gck</i> produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and β-type. Steady-state viscosity variation studies indicate that the <i>k</i>_cat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the <i>k</i>_cat value of a β-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the β-type variant lacks a burst phase. Comparative hydrogen–deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active site loop, which folds upon binding of glucose, is protected from exchange in the α-type variant. The α-type variant also displays an increased level of exchange within a β-strand located near the enzyme’s hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, β-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas β-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK’s unique kinetic cooperativity

Algae Polar Lipids Characterized by Online Liquid Chromatography Coupled with Hybrid Linear Quadrupole Ion Trap/Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

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, <i>Nannochloropsis oculata</i>, 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

Positive Ion Electrospray Ionization Suppression in Petroleum and Complex Mixtures

Since the emergence of high resolving power crude oil mass spectrometry two decades ago, hundreds of publications and presentations have detailed petroleum complex mixtures by electrospray ionization (ESI) mass spectrometry (MS). None of these works have reported or detailed ion suppression (also referred to as ionization biasing or matrix effects) which is a well-known feature of ESI. Here, we show the extreme consequences of ionization biasing within a narrow, 1 order of magnitude concentration range for crude oil mixture direct infusion experiments in positive ion (+) ESI. An oil spill contaminant, a crude oil, and an equimolar model compound mixture were electrosprayed at various analyte and acid modifier concentrations for Fourier transform ion cyclotron resonance (FT-ICR) and time-of-flight (TOF) MS analysis. A 3-fold increase in the number of elemental compositions is achieved by optimization of analyte and acid concentration. At high analyte concentration, oxygen heteroatom class (i.e., C_cH_hO_<i>x</i> species, denoted henceforth simply as O_<i>x</i>) abundance is attenuated and practically nullified. The suppression can be understood from (+) ESI TOF mass analysis of a prepared equimolar model compound mixture, particularly those with ketone functional groups. At sufficiently low concentration of analyte, the relative abundances of nitrogen- and oxygen-containing model compounds no longer vary. For (+) ESI at the flow rates and voltages described in this study, we recommend operating at mass/volume petroleum residue concentration below 0.1 mg/mL in 1:1 (v:v) toluene/methanol with formic acid at 2.5% (v:v)

Advanced Chemical Characterization of Pyrolysis Oils from Landfill Waste, Recycled Plastics, and Forestry Residue

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

Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry

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 C_H2 and C_H3, and the disulfide bond (S–S)-linked heavy-chain constant domain C_H3. 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 C_H1 and C_H2 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