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Brassinosteroid (BR) regulates diverse physiological and developmental processes in plants (Choudhary et al., 2012). BR binds to the receptor kinase BRI1 to trigger a phosphorylation/dephosphorylation cascade, which includes phosphorylation of Brassinosteroid Signaling Kinase 1 (BSK1) and Constitutive Differential Growth 1 (CDG1) by BRI1, phosphorylation of BSU1 by CDG1, and dephosphorylation of BIN2 (a GSK3-like kinase) by BSU1 (Kim et al., 2009, 2011; Wang et al., 2014). Almost all the downstream components of BR signaling are encoded by multiple genes of a family.

Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or “degrons”) contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.

The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This resulted in the identification of PTM configurations supported by unique fragment ions, and their relative abundances were estimated by use of a non-negative least squares procedure.

The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we have performed a proteomic study of BR-regulated proteins in Arabidopsis using two-dimensional difference gel electrophoresis (2-D DIGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR insensitive mutant bri1-116 and BR hypersensitive mutant bzr1-1D identified 5 proteins (PATL1, PATL2, THI1, AtMDAR3 and NADP-ME2) affected by both BR-treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knockout mutants or immunoblotting. Interestingly, about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein- and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore, BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in 2-D DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.

Combinatorial methodologies are emerging as powerful tools in heterogeneous catalysis. Previously, we have established a strong correlation between catalysts prepared as thin films for high-throughput primary screening to those prepared in bulk for secondary screening in a multi-channel fixed bed (MCFB) microreactor for the ethane oxidative dehydrogenation process [Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 11077]. Numerous catalysts with improved performance over literature compositions were also reported. With this high-throughput technology, tens of thousands of new compositions have been synthesized and tested. In this paper, we describe in detail the discovery of a new class of catalysts with extraordinary performance improvements. These catalysts contain Ni, Nb, and Ta or Co mixed oxides. The respective ternary systems were first screened in a thin film form (∼200 μg catalyst loading). Then they were scaled up to 50 mg level and screened in an MCFB microreactor. The optimal compositions were Ni0.62Ta0.10Nb0.28Ox and Ni0.62Ta0.20Nb0.18Ox. They convert 20.5% of ethane with 86.2% selectivity to ethylene and 20.0% of ethane with 86.1% selectivity to ethylene at 300 °C, respectively, as compared to 3.3% conversion of ethane with 82.9% selectivity to ethylene for the state-of-the-art catalyst Mo0.72V0.26Nb0.02Ox under identical testing conditions. One of these compositions, Ni0.62Ta0.10Nb0.28Ox, was further scaled up to 5 g and tested in a conventional bench scale reactor. The performance improvement is consistent with measurements made on the MCFB microreactor.

High-throughput primary synthesis and screening methods have been applied to the heterogeneously-catalyzed gas phase oxidation of ethane to acetic acid using mixed metal oxide catalysts. The discovery libraries consisted of 16×16 arrays of 256 catalysts on 3″×3″ quartz wafers. Catalysts were prepared using automated liquid-dispensing techniques and screened in parallel for catalytic activity in a Symyx Technologies 256-channel microfluidic reactor. Product detection was performed using parallel colorimetric techniques on products adsorbed on silica-coated glass TLC plates. This workflow allows the screening of more than 3000 samples per day. Promising leads were confirmed in focus libraries and are being optimized in secondary screening. MoV was identified as the most active binary of redox metals and was subsequently doped with main group, rare earth, and transition metals to form ternaries. Prior art MoVX (X=Nb, Ni, Sb) catalysts were successfully reproduced and it was shown that Pd doping significantly increases the catalytic activity of these systems. Three novel MoVY ternary systems were discovered.

High-throughput synthesis and screening methods have been developed for the direct amination of benzene to aniline using solid cataloreactants as oxidants. The discovery libraries consisted of hundreds of samples in the primary screen, and arrays of 24 catalysts in the secondary screen. Catalysts were prepared in multi-well batch reactors and screened in parallel for catalytic activity using modified TLC detection as a primary screen and fast serial GC detection as a secondary screen. Around 25,000 samples were screened in about a year. Promising hits identified in the high-throughput screens were successfully scaled up and optimized in conventional autoclaves. Novel cataloreactant systems consisting primarily of a noble metal and a reducible metal oxide have been discovered. Rh, Ir, Pd, and Ru were found to be suitable noble metal dopants. Ni and Co oxides were the only active and selective oxidants identified. Ni is the most active oxidant but requires a Mn dopant as a stabilizer to improve the regenerability, whereas Co is stable even at high regeneration temperatures. The best performing cataloreactants contain Rh or Ir as noble metals, NiO as an oxidant, and ZrO 2 or K–TiO 2 as carrier. An optimized cataloreactant, Rh/Ni–Mn/K–TiO 2 achieved stable 10% benzene conversion and >95% selectivity to aniline at 300 °C and 300 bar. Significantly, the cataloxidant can be regenerated repeatedly without a substantial loss of performance by reoxidation in air. Any noble metal oxides formed during regeneration are effectively reduced in situ during the next amination reaction.

The decay amplitude envelope of an ICR time-domain signal determines its corresponding Fourier transform mass spectral line shape. The commonly accepted FT-ICR frequency-domain unapodized Lorentzian spectral line shape originates from the Langevin ion-neutral collision model, in which an ion is treated as a point charge that induces an electric dipole moment in a neutral collision partner. The Langevin model provides a good description of reactions of low-energy collisions of low-mass positive ions with neutrals. However, the Langevin model is inappropriate for collisions of high-mass gas-phase biopolymer ions with low-mass neutrals. Here, we examine ion trajectories for both Langevin and hard-sphere ion-neutral collision models. For the Langevin model, collision frequency is independent of ion speed, leading to a linear differential equation of ion motion with a frictional damping term linearly proportional to ion velocity. For the hard-sphere model, collision frequency is proportional to ion speed and the frictional damping term is proportional to the square of ion velocity. We show that the resulting (non-linear) equation of ion motion leads to a non-exponential time-domain ICR signal whose amplitude envelope has the form, 1 (1 + σt) , in which σ is a constant. Dispersion-vs-absorption (DISPA) line shape analysis reveals that the ‘hard-sphere’ spectral line shape resembles that of overlaid narrow and broad Lorentzians. We discuss several important implications of the new ‘hard-sphere’ line shape for ICR spectral analysis, ICR signal processing, collision-based ion activation, and ion axialization. Finally, in the hard-sphere limit, a non-linear frictional damping term will also apply to ions in a Paul trap.

Stored waveform excitation produced by inverse Fourier transformation of a specified magnitude/phase excitation spectrum offers the most general and versatile means for broadband mass-selective excitation and ejection in Penning (FT-ICR) and Paul (quadrupole) ion trap mass spectrometry. Since the last comprehensive review of SWIFT excitation in 1987, the technique has been adopted, modified, and extended widely in both the ICR and quadrupole ion trap communities. Here, we review the principles, variations, algorithms, hardware implementation, and some applications of SWIFT for both ICR and quadrupole ion trap mass spectrometry. We show that the most desirable SWIFT waveform is that optimized to reduce both the time-domain SWIFT maximum amplitude and the amplitude near the start and end of the SWIFT waveform. We examine the “true” magnitude excitation spectrum, obtained by zero-filling and forward Fourier transforming the SWIFT time-domain waveform, in order to evaluate the trade-off between spectral magnitude uniformity and frequency (mass) selectivity. Apodization of the SWIFT waveform is optimally conducted by smoothing the excitation magnitude spectrum prior to generation of the SWIFT waveform by inverse FT. When (as for broadband ejection in a quadrupole ion trap) it is important that ions be excited near-simultaneously over a wide mass range, the phase spectrum (before inverse FT to generate the SWIFT waveform) may be overmodulated or randomly modulated (“filtered noise field”), with the recognition that very substantial non-uniformity in the “true” excitation magnitude spectrum will result.

This paper offers a brief introduction to the ion optics of FT-ICR mass spectrometry, followed by selected examples of various means for improved simulation and control of ion trajectories in a Penning ion trap (which may be of hyperboloidal, cylindrical, or cubic configuration), as well as means for injecting ions through a magnetic field gradient by use of an rf-only octupole ion guide.

Virtually all Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry experiments are based on ion confinement in a Penning trap: namely, a spatially homogeneous static magnetic field B, and a three-dimensional axial (along B) quadrupolar electrostatic trapping potential. In addition, it is desirable to provide an alternating azimuthal (i.e. in a plane perpendicular to B) spatially uniform dipolar electric potential to generate or detect ion cyclotron coherence and an alternating azimuthal quadrupolar potential in the presence of collisional cooling to axialize ions. In this paper, we show how each of these three potentials may be generated by infinitely extended electrode arrangements. We then examine the potentials actually produced by various (finite size) ICR ion trap geometric configurations. Finally, we show that by segmenting the electrodes of an ICR ion trap, and applying an appropriate potential to each segment, near-perfect potentials of all three types may be generated. The necessary principles and tools (Laplace's equation: harmonic potential; reciprocity theorem; V-vector method) are presented, followed by derivation and/or description and rationale for all of the principal ICR ion trap designs. We close by proposing a new “universal” trap consisting of six planar grids arranged in a cube: such a configuration is the first to promise near-perfect potentials of all three types while maintaining the symmetry and compact shape of a cubic Penning trap.

Highly efficient ion injection into an ICR ion trap of ions generated outside the magnetic field is crucial for successful coupling of a high pressure external ion source, such as electrospray ionization, to an FT-ICR mass spectrometer. Any of several ion transport systems, including r.f.-only quadrupole, r.f.-only octupole, electrostatic einzel lenses, electrostatic ion guide, or use of a supersonic jet expansion to ensure that ions remain virtually on-axis during injection, can overcome the magnetic mirror effect and transport ions at high pressure through a magnetic field gradient toward a low pressure ICR ion trap. However, ions thus transported may exhibit a wide distribution in kinetic energy, making it difficult to trap them all. Gated trapping and pulsed-gas methods are useful for trapping ions from a pulsed source (e.g. laser desorption/ionization), but are not efficient for trapping of continuously injected ions (e.g. electrospray). Here, we introduce a novel method for trapping a continuously generated beam of ions, by injecting ions through a port held at a potential lower than that of the axial front trapping electrode and displaced laterally from the symmetry axis of the trap. Ions reflected from the back trap plate undergo magnetron rotation which in general prevents the ions from exiting back through their injection point. Ion/neutral collisions damp axial motion, and further reduce the ion's chance of escape. Supercomputer simulations of ion trajectories in the three-dimensional electrostatic potential, with collisions modeled as a frictional resistive force, predict efficient trapping of continuously injected ions up to mass-to-charge ratio m/z 50 000 over a wide range (>7 eV) of ion kinetic energy.

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has been used to determine the masses of the stable neon isotopes, 20 Ne and 22 Ne. From the mass difference, m ( 2 H 2 16 O + ) − m ( 20 Ne + ) = 0.030 677 480(67) u and previous accurate measurements of the masses of 16 O and 1 H, we have determined the atomic mass of 20 Ne as 19.992 440 691 (90) u. From the measured mass difference, m ( 22 Ne + − m ( 20 Ne + ) = 1.998 943 585(290) u, the atomic mass of 22 Ne has been calculated as 21.991 384 276(304) u. Both of these newly determined isotopic masses are significantly more precise (factor of 24 for 20 Ne and factor of 6 for 22 Ne), and deviate significantly (e.g. by more than 2σ accepted for 20 Ne) relative to the accepted values in the 1983 Atomic Mass Evaluation (based on nuclear reaction data), but are in excellent agreement with prior (less precise) magnetic sector-based mass-spectrometric measurements of these masses. The calculated two-neutron separation energy for 22 Ne/ 20 Ne is 17 126 662(270) eV.

A segmented cubic ion cyclotron resonance (ICR) ion trap configuration that generates an r.f. electric excitation potential of approximately xz (two-dimensional quadrupolar) symmetry near the center of the trap is proposed. A new ion excitation/detection scheme is based on the analysis of image current induced by the combination of axial and azimuthal (cyclotron and magnetron) ion motions on the same electrode configuration. Ions may thereby be resonantly excited or detected at each of the combination frequencies ωz + ω−, ωz − ω−, ω+ + ωz, and ω+ − ωz, in which ωz is the axial (i.e. z-motion) frequency, ω− is the magnetron frequency, and ω+ is the reduced cyclotron frequency. The trajectory of an ion subjected to resonant xz excitation is derived analytically with provision for collisional damping. For example, when ions are excited at the frequency ωz + ω−, magnetron motion is periodically interconverted into axial motion. The coupling of magnetron and axial motions in presence of ion/neutral collisions (treated analytically as a frictional damping force) provides a method for axialization and cooling of heavy ions. Conditions for such coupling and cooling are analyzed in detail: under representative experimental conditions, magnetron-to-axial conversion may be achieved by xz quadrupolar excitation amplitude of just a few millivolts. Combination of the present method and previously demonstrated azimuthal (xy) quadrupolar excitation should result in both axial and radial compression of an ion cloud for improved Fourier transform ICR sensitivity and mass resolution.

A quadrature method for producing circularly polarized excitation for selective detection of positive or negative ions has been proposed for Fourier-transform ion cyclotron resonance mass spectrometry. The new technique achieves the same post-excitation ion cyclotron orbital radius at half the excitation rf voltage of conventional linearly polarized (dipolar) excitation, and is especially useful for exciting and detecting dynamically trapped ions of one charge state in a mixture of simultaneously trapped positive and negative ions. Circularly polarized single-frequency resonant excitation is accomplished experimentally by simultaneous application ofa sinusoidal rf voltage to one pair of electrodes of a cubic ICR ion trap and a 90° or 270° phase-shifted voltage of the same frequency to a second orthogonal pair of electrodes, followed by conventional dipolar detection. Selective excitation of 35C1+ or 35CI− ions from a dynamically trapped mixture of 35C1+ and 35C1− ions is demonstrated. Circularly polarized broadb excitation by use of frequency-sweep or stored-waveform (SWIFT) excitation is proposed and discussed.

A recently developed technique for axialization of ions in a Penning (i.e. hyperbolic) trap has been adapted for Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry in a dual cubic trap instrument. A resonant alternating electric azimuthal quadrupolar field at the combination frequency, ωc = ω+ + ω−, converts ion magnetron motion into collision-damped cyclotron motion. The usual collision- induced drift of ions from the center of the trap under the influence of the radially outward-directed electric force of the trapping field is thereby effectively inverted, so that ions with off-axis ICR orbit centers may be driven back to on-axis low radius ion cyclotron orbits. This effect is frequency-dependent and therefore mass-selective. The experiment requires only minor wiring modifications to an existing cubic, tetragonal, or cylindrical ICR ion trap. With this technique, we have been able to store benzene molecular ions for several seconds at high pressure (> 1 x 10−5 mbar) in one half of a dual trap, as demonstrated by subsequent transfer of these ions to the other half of the dual trap for ICR excitation and detection. We also report simultaneous axialization and extended trapping of ions of different mass-to-charge ratios by use of multiple-frequency stored-waveform inverse Fourier transform excitation. Proposed applications include axialization and cooling for improved detection of product ions in MSMS experiments, ion trapping at high pressure for extended periods for measurement of slow ion/molecule reaction rates, and increased sensitivity for detection of externally injected ions.

CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation.