Ruthenium versus platinum: interactions of anticancer metallodrugs with duplex oligonucleotides characterised by electrospray ionisation mass spectrometry

The binding of the ruthenium-based anticancer drug candidates KP1019, NAMI-A and RAPTA-T towards different double-stranded oligonucleotides was probed by electrospray ionisation mass spectrometry and compared with that of the widely used platinum-based chemotherapeutics cisplatin, carboplatin and oxaliplatin. It was found that the extent of adduct formation decreased in the following order: cisplatin > oxaliplatin > NAMI-A > RAPTA-T > carboplatin > KP1019. In addition to the characterisation of the adducts formed with the DNA models, the binding sites of the metallodrugs on the oligonucleotides were elucidated employing top-down tandem mass spectrometry and were found to be similar for all the metallodrugs studied, irrespective of the sequence of the oligonucleotide. A strong preference for guanine residues was established.     

Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells

In this study, a recombinant monoclonal IgG antibody was produced by transient gene expression (TGE) in suspension-adapted HEK-293E cells. The objective of the study was to determine the variation in recombinant IgG yield and glycosylation in ten independent transfections. In a ten-day batch process, the variation in transient IgG yield in the ten batches was less than 30% with the specific productivity averaging 20.2 ± 2.6 pg/cell/day. We characterized the N-glycosylation profile of each batch of affinity-purified IgG by intact protein and bottom-up mass spectrometry. Four major glycans were identified at Asn(297) in the ten batches with the maximum relative deviation for a single glycoform being 2.5%. In addition, within any single transfection there was little variation in glycoforms over the ten-day culture. Our experimental data indicate that with TGE, the production of recombinant IgG with little batch-to-batch variation in volumetric yield and protein glycosylation is feasible, even in a non-instrumented cultivation system as described here.     

Comparative evaluation of pectolytic and proteolytic enzyme production by free and immobilized cells of some strains of the phytopathogenic Erwinia chrysanthemi

Whole cells of the phytopathogenic Erwinia chrysanthemi strains were immobilized in k-carrageenan and grown in high-calcium Xanthomonas campestris medium containing sodium polypectate as carbon source. All the strains used survived immobilization into k-carrageenan beads. Immobilized E. chrysanthemi strains displayed higher pectolytic and proteolytic enzyme activities than free cells in liquid suspension. Carrageenan immobilization techniques could provide a system to mimic the conditions of E. chrysanthemi cells in the infected plant tissue. This could prompt a thorough study of the factors governing the biosynthesis of virulence factors by this bacterium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 215–219. Received 04 April 2001/ Accepted in revised form 12 June 2001     

Light intensity regulates growth and reproduction of a snail grazer (Gyraulus chinensis) through changes in the quality and biomass of stream periphyton

1. The light : nutrient hypothesis (LNH) proposes that herbivore growth rates are maximised at intermediate light‐to‐nutrient ratios. A reduction to light intensity (i.e. decreased light‐to‐nutrient ratio) should lead to reduced food availability for herbivores while excessive light intensity in oligotrophic environments (i.e. increased light‐to‐nutrient ratios) should increase the C : N and C : P ratios of producers. However, this hypothesis has not yet been supported by studies on stream ecosystems. 2. We tested the LNH by experimental application of controlled natural gradients in light intensity to oligotrophic laboratory channels that included periphyton and the freshwater snail Gyraulus chinensis. 3. The results in this oligotrophic environment indicate that light regulated the flow of matter between trophic levels and grazer reproduction by controlling C : P ratios of the producers.     

The ecology and agronomy of Miscanthus sinensis, a species important to bioenergy crop development, in its native range in Japan: a review

Among several candidate perennial taxa, Miscanthus × giganteus has been evaluated and promoted as a promising bioenergy crop. Owing to several limitations, however, of the sterile hybrid, both at the taxon and agronomic production levels, other options need to be explored to not only improve M . × giganteus , which was originally collected in Japan, but to also consider the development of other members of its genus, including Miscanthus sinensis , as bioenergy crops. Indeed, there is likely much to be learned and applied to Miscanthus as a bioenergy crop from the long history of intensive interaction between humans and M. sinensis in Japan, which in some regions of the country spans several thousand years. Combined with its high amount of genetic variation, stress tolerance, biotic interactions with fauna, and function as a keystone species in diverse grasslands and other ecosystems within its native range, the unique and extensive management of M. sinensis in Japan as a forage grass and building material provides agronomists, agroecologists, and plant breeders with the capability of better understanding this species in terms of potential contribution to bioenergy crop development. Moreover, the studies described in this review may serve as a platform for future research of Miscanthus as a bioenergy crop in other parts of the world.     

Aboveground plant biomass,carbon, and nitrogen dynamics before and after burning in a seminatural grassland of Miscanthus sinensis in Kumamoto,Japan

Although fire has been used for several thousand years to maintain Miscanthus sinensis grasslands in Japan, there is little information about the nutrient dynamics in these ecosystems immediately after burning. We investigated the loss of aboveground biomass; carbon (C) and nitrogen (N) dynamics; surface soil C change before and after burning; and carbon dioxide (CO₂), methane (CH₄), and nitrous oxide (N₂O) fluxes 2 h after burning in a M. sinensis grassland in Kumamoto, Japan. We calculated average C and N accumulation rates within the soil profile over the past 7300 years, which were 58.0 kg C ha^?1 yr^?1 and 2.60 kg N ha^?1 yr^?1, respectively. After burning, 98% of aboveground biomass and litter were consumed. Carbon remaining on the field, however, was 102 kg C ha^?1. We found at least 43% of C was possibly lost due to decomposition. However, remaining C, which contained ash and charcoal, appeared to contribute to C accumulation in soil. There was no difference in the amount of 0–5 cm surface soil C before and after burning. The amount of remaining litter on the soil surface indicated burning appeared not to have caused a reduction in soil C nor did it negatively impact the sub‐surface vegetative crown of M. sinensis. Also, nearly 50 kg N ha^?1 of total aboveground biomass and litter N was lost due to burning. Compared with before the burning event, postburning CO₂ and CH₄ fluxes from soil appeared not to be directly affected by burning. However, it appears the short time span of measurements of N₂O flux after burning sufficiently characterized the pattern of increasing N₂O fluxes immediately after burning. These findings indicate burning did not cause significant reductions in soil C nor did it result in elevated CO₂ and CH₄ emissions from the soil relative to before the burning event.     

Micro-capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry--a powerful tool for peptide and protein identification

In order to be able to study complex biological samples, a micro-capillary liquid chromatography system was coupled to a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer. The setup was tested on a tryptic digest of bovine serum albumin, which resulted in high sequence coverage (> 92%) of the protein.     

Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure–function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.Top-down mass spectrometry (MS)¹ (1–3) has continued to demonstrate its particular advantages over traditionally employed bottom-up MS strategies (4). Specifically, top-down MS allows the characterization of specific protein isoforms originating from the alternative splicing of mRNA that code single nucleotide polymorphisms and/or post-translational modifications (PTMs) of protein species (5). Intact protein molecular weight (MW) determination and subsequent gas-phase fragmentation of selected multiply charged protein ions (referred to as tandem MS or MS/MS) theoretically might result in complete protein sequence coverage and precise assignment of the type and position of PTMs, amino acid substitutions, and C- or N-terminal truncations (6), whereas the bottom-up MS approach allows only the identification of a certain protein family when few or redundant peptides are found for a particular protein isoform. At a practical level, however, top-down MS-based proteomics struggles not only with the single- or multi-dimensional separation of undigested proteins, which demonstrates lower reproducibility and repeatability than for peptides, but also with technical limitations present in even state-of-the-art mass spectrometers. The outcome of a top-down MS experiment depends indeed on the balance between the applied resolution of the mass spectrometer and its sensitivity. The former is required for unambiguous assignment of ion isotopic clusters in both survey and MS/MS scans, whereas the latter is ultimately dependent on the scan speed of the mass analyzer, which determines the number of scans that can be accumulated for a given analyte ion on the liquid chromatography (LC) timescale to enhance the resulting signal-to-noise ratio (SNR). Until recently, the instrument of choice for top-down MS has been the Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, primarily because of its superior resolving power and the availability of electron capture dissociation for the efficient MS/MS of large biomolecules (7, 8). However, this solution has been shown to have some limitations in the analysis of large proteins (9). The main issue, as described by Compton et al. (10), is that the SNR in Fourier transform mass spectrometry (FTMS) is inversely proportional to the width of the isotopic and charge state distributions (11), which both increase as a function of MW. Particularly, the SNR dramatically decreases with MW under standard on-line LC-MS/MS operating conditions if isotopic resolution is required. It is noteworthy that such SNR reduction can affect not only intact mass measurements, but also the subsequent MS/MS performance.The most widely employed solution for improving top-down analysis is thus a substantial reduction of the protein mixture complexity, for example, through off-line sample prefractionation (12). Furthermore, when the MW exceeds 100 kDa, proteins are often analyzed via direct infusion after off-line purification of the single isoform or species of interest (13). Overall, these strategies aim to improve the quality of mass spectra, specifically their SNR, by increasing the number of scans dedicated to each selected isoform or species. However, off-line intact protein analysis has limitations, including sample degradation and modification (e.g., oxidation during long off-line measurements and sample storage). The time required for multistep LC-based protein purification can also be substantial.Electron capture dissociation (ECD) (14, 15) and electron transfer dissociation (ETD) (16) are ion activation techniques that allow polypeptide fragmentation with reduced PTM losses (17, 18). Nevertheless, ECD and ETD generally provide larger sequence coverage for intact proteins than slow-heating activation methods such as collision induced dissociation (CID) and infrared multiple photon dissociation (19, 20). Furthermore, ECD and ETD are known to cleave disulfide bonds, a fundamental feature for the analysis of proteins in their native state (i.e., without cysteine reduction and alkylation) (21–23).The structural analysis of high MW intact proteins with MS has garnered much recent attention in the literature (24, 25), mainly because of the improved capabilities offered by rapidly developing sample preparation, protein separation, and mass spectrometric methods and techniques. Immunoglobulin G (IgG) proteins are antibodies with an MW of about 150 kDa that are composed of two identical sets of light and glycosylated heavy chains with both intra- and intermolecular disulfide bridges (Fig. 1) (26). IgGs represent an attractive target for structural analysis method development, given their high importance as biotherapeutics (27). A unit-mass resolution mass spectrum demonstrating an isotopic distribution of an isolated charge state of a 148 kDa IgG1 has been recently achieved with FT-ICR MS equipped with 9.4 T superconducting magnet and a statically harmonized ICR cell (24). However, further analytical improvements are needed to achieve routine and reproducible MS operation at the required level of resolution and sensitivity.Open in a separate windowFig. 1.Schematic representation of IgG1. Two identical light (blue) and two identical heavy (fucsia) chains form the intact IgG. The light chain is composed of a variable domain (V_L) and a constant domain (C_L), whereas the heavy chain comprises one variable domain (V_H) and three constant domains (C_H1–3). Each domain contains an intramolecular disulfide bridge (in red); intermolecular disulfide bridges link the heavy chains to each other (two bonds) and each heavy chain to one light chain (one bond). Each heavy chain includes an N-glycosylation site (located at Asn²⁹⁷; here, a G0F/G0F glycosylation is shown).Fragmentation of intact antibodies in the gas phase following the top-down MS approach has been previously attempted without precursor ion charge state isolation by means of nozzle-skimmer CID on a linear trap quadrupole (LTQ)-Orbitrap™ (28, 29) and with precursor ion isolation via ETD on a high resolution quadrupole time-of-flight (qTOF) mass spectrometer (25). Relative to the results previously obtained with slow-heating MS/MS methods, the ETD qTOF MS/MS demonstrated substantially higher sequence coverage, reaching 15% for human and 21% for murine IgGs. Important for future top-down proteomics development for complex protein mixtures, the ETD qTOF MS/MS results were obtained on the LC timescale. To increase the sequence coverage and confidence in product ion assignment, a substantial increase in SNR was achieved by averaging MS/MS data from up to 10 identical LC-MS/MS experiments. The high complexity of the product ion population reduced the effective resolution to about 30,000, presumably limiting the assignment of overlapping high charge state product ions in the 1000–2000 m/z range. Even higher peak complexity was observed in the region of charge reduced species and complementary heavy product ions, above 3000 m/z. Finally, numerous disulfide bonds drastically reduced MS/MS efficiency in the disulfide bond-protected regions.Here we demonstrate that ETD-enabled hybrid linear ion trap Orbitrap FTMS allows us to further improve the top-down ETD-based LC-MS/MS of monoclonal antibodies, introduced earlier for TOF-based MS. To fully take advantage of the high resolving power of Orbitrap MS/MS for increasing both the number of assigned product ions and the confidence of the assignments, maintaining an LC-MS/MS setup useful in a general proteomics workflow for protein desalting and separation, we averaged time-domain transients (derived from separated LC-MS/MS runs) before Fourier transform signal processing.     

Stability of an organometallic ruthenium-ubiquitin adduct in the presence of glutathione: relevance to antitumour activity

The interactions of the ruthenium(II) complex Ru(η⁶-p-cymene)(pta)Cl₂ (RAPTA-C), an effective anticancer and antimetastatic agent, with biological nucleophiles are important with respect to its mechanism of action, for example, the reaction with glutathione (GSH) potentially plays an important role in detoxification. RAPTA-C reacts rapidly with glutathione forming a series of adducts including Ru(η⁶-p-cymene)(pta)(GS), Ru(η⁶-p-cymene)(GS) and bis-GSH conjugates, which were characterised by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In addition, the ability of glutathione to cleave ruthenium-ubiquitin bonds was assayed and it was shown that GSH is capable of removing the Ru moiety from the protein, although no ternary adducts were identified.     

Carbon budget and methane and nitrous oxide emissions over the growing season in a Miscanthus sinensis grassland in Tomakomai,Hokkaido, Japan

Species in the Miscanthus genus have been proposed as biofuel crops that have potential to mitigate elevated atmospheric carbon dioxide (CO₂) levels and nitrous oxide (N₂O) and methane (CH₄) emissions. Miscanthus sinensis is widespread throughout Japan and has been used for biomass production for centuries. We assessed the carbon (C) budget and N₂O and CH₄ emissions over the growing season for 2 years in a M. sinensis‐dominated grassland that was naturally established around 1972 in Tomakomai, Hokkaido, Japan, which is near the northern limit for M. sinensis grassland establishment on Andisols. Average C budget was ?0.31 Mg C ha^?1, which indicates C was released from the grassland ecosystem to the atmosphere. Dominant components in the C budget appeared to be aboveground net primary production of plants (1.94–2.80 Mg C ha^?1) and heterotrophic respiration (2.27–3.11 Mg C ha^?1). The measurement of belowground net primary production (BNPP) of plants in the M. sinensis grassland was extremely variable, thus only an approximate value could be calculated. Mean C budget calculated with the approximated BNPP value was 1.47 and ?0.23 Mg C ha^?1 for 2008 and 2009, respectively. Given belowground biomass (9.46–9.86 Mg C ha^?1) was 3.1–6.5 times higher than that of aboveground biomass may provide additional evidence suggesting this grassland represents a C sink. Average CH₄ emissions across years of ?1.34 kg C ha^?1 would indicate this grassland acts as an atmospheric CH₄ sink. Furthermore, average N₂O emissions across years were 0.22 kg N ha^?1. While the site may contribute N₂O to the atmosphere, this value is lower compared with other grassland types. Global warming potential calculated with the approximated BNPP value was ?5.40 and 0.95 Mg CO₂ Eq ha^?1 for 2008 and 2009, respectively, and indicates this grassland could contribute to mitigation of global warming.