Mass spectrometry imaging of glucosinolates in arabidopsis flowers and siliques

Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation

Nanoelectronic thermometers optimised for sub-10 millikelvin operation

We report the cooling of electrons in nanoelectronic Coulomb blockade thermometers below 4 mK. Above 7 mK the devices are in good thermal contact with the environment, well isolated from electrical noise, and not susceptible to self-heating. This is attributed to an optimised design that incorporates cooling fins with a high electron-phonon coupling and on-chip electronic filters, combined with a low-noise electronic measurement setup. Below 7 mK the electron temperature is seen to diverge from the ambient temperature. By immersing a Coulomb Blockade Thermometer in the 3He/4He refrigerant of a dilution refrigerator, we measure a lowest electron temperature of 3.7 mK.Comment: 11 pages, 4 figures. (Fixed fitted saturation T_e on p9

Monitoring recombinant protein expression in bacteria by rapid evaporative ionisation mass spectrometry.

RATIONALE:There is increasing interest in methods of direct analysis mass spectrometry that bypass complex sample preparation steps. METHODS:One of the most interesting new ionisation methods is rapid evaporative ionisation mass spectrometry (REIMS) in which samples are vapourised and the combustion products are subsequently ionised and analysed by mass spectrometry (Synapt G2si). The only sample preparation required is the recovery of a cell pellet from a culture that can be analysed immediately. RESULTS:We demonstrate that REIMS can be used to monitor the expression of heterologous recombinant proteins in Escherichia coli. Clear segregation was achievable between bacteria harvesting plasmids that were strongly expressed and other cultures in which the plasmid did not result in the expression of large amounts of recombinant product. CONCLUSIONS:REIMS has considerable potential as a near-instantaneous monitoring tool for protein production in a biotechnology environment

Top-down and bottom-up identification of proteins by liquid extraction surface analysis mass spectrometry of healthy and diseased human liver tissue

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15–25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-014-0967-z) contains supplementary material, which is available to authorized users

Rapid identification of species, sex and maturity by mass spectrometric analysis of animal faeces

Background We describe a new approach to the recovery of information from faecal samples, based on the analysis of the molecular signature generated by rapid evaporative ionisation mass spectrometry (REIMS). Results Faecal pellets from five different rodent species were analysed by REIMS, and complex mass spectra were acquired rapidly (typically a few seconds per sample). The uninterpreted mass spectra (signatures) were then used to seed linear discriminant analysis and classification models based on random forests. It was possible to classify each species of origin with a high rate of accuracy, whether faeces were from animals maintained under standard laboratory conditions or wild-caught. REIMS signatures were stable to prior storage of the faecal material under a range of different conditions and were not altered rapidly or radically by changes in diet. Further, within species, REIMS signatures could be used to discriminate faeces from adult versus juvenile mice, male versus female mice and those from three different laboratory strains. Conclusions REIMS offers a completely novel method for the rapid analysis of faecal samples, extending faecal analysis (previously focused on DNA) to an assessment of phenotype, and has considerable potential as a new tool in the armamentarium of the field biologist

Exploring the conformational landscape and stability of Aurora A using ion-mobility mass spectrometry and molecular modelling

ABSTRACTProtein kinase inhibitors are proving highly effective in helping treat a number of non-communicable diseases driven by aberrant kinase signaling. They are also extremely valuable as chemical tools to help delineate cellular roles of kinase signaling complexes. The binding of small molecule inhibitors induces conformational effects on kinase dynamics; evaluating the effect of such interactions can assist in developing specific inhibitors and is deemed imperative to understand both inhibition and resistance mechanisms. Using gas-phase ion mobility-mass spectrometry (IM-MS) we characterized changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Aided by molecular modeling, we establish three major conformations: one highly-populated compact conformer similar to that observed in most crystal structures, a second highly-populated conformer possessing a more open structure that is infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank. Comparison of active (phosphorylated) and inactive (non-phosphorylated) forms of Aur A revealed that the active enzyme has different conformer weightings and is less stable than the inactive enzyme. Notably, inhibitor binding shifts conformer balance towards the more compact configurations adopted by the unbound enzyme, with both IM-MS and modelling revealing inhibitor-mediated stabilisation of active Aur A. These data highlight the power of IM-MS in combination with molecular dynamics simulations to probe and compare protein kinase structural dynamics that arise due to differences in activity and as a result of compound binding.</jats:p

Utilising biological geotextiles: Introduction to the BORASSUS project and global perspectives

Field and laboratory studies indicate that utilisation of biological geotextiles constructed from palm-leaves and other selected organic materials are an effective, sustainable and economically viable soil conservation technique. The three-year plus (1 July 2005–28 February 2009) EU-funded BORASSUS Project (contract no. INCO-CT-2005-510745) evaluated the long-term effectiveness of biological geotextiles in controlling soil erosion and assessing their sustainability and economic viability. These studies progressed in ten countries, both in the ‘industrial north’ (in Europe) and in the ‘developing south’ (Africa, South America and South East Asia). The studied countries in the ‘developing south’ included Brazil, China, The Gambia, South Africa, Thailand and Vietnam. The ‘industrial north’ countries included Belgium, Hungary, Lithuania and the UK. The main findings of these studies are summarised in this paper and thematic information is presented in the other four papers in this Special Issue. Biological geotextiles offer potentially novel bioengineering solutions to environmental problems, including technologies for soil conservation, sustainable plant production and use of indigenous plants, improved ecosystem management by decreasing deforestation, improving agroforestry and cost-effective biogeotextile applications in diverse environments. Biogeotextiles may provide socio-economic platforms for sustainable development and the benefits for developing countries may include poverty alleviation, engagement of local people as stakeholders, employment for disadvantaged groups, small and medium enterprise (SME) development, earning hard currency, environmental education and local community involvement in land reclamation and environmental education programmes. These benefits are achieved through: (i) promotion of sustainable and environmentally friendly palm-agriculture to discourage deforestation, promoting both reforestation and agroforestry; (ii) construction of biogeotextiles enabling development of a rural labour-intensive industry, particularly encouraging employment of socially disadvantaged groups and (iii) export of biogeotextiles to industrialised countries could earn hard currency for developing economies, based on the principles of fair trade. Research and development activities of the BORASSUS Project have improved our knowledge on the effect of biogeotextile mats on the micro- and macro-soil environments and at larger scales through controlled laboratory and field experiments in diverse environments