369 research outputs found
Towards chemical profiling at the cellular level
Traditional methods for the analysis of cellular components have focused on 'grid
and find' assays that provide quantitative information from a large population of
cells, often as many as a million cells. The results of these studies are often
presented only as the percentage of the dry weight of the cells and not the
concentration within individual cells. The research presented in this thesis is
concerned with the development and application of methods for single cell sampling
and analysis (SiCSA) from fungal cells that overcomes this deficit. The methods
developed offer the potential to investigate the intra-cellular concentration of
biologically relevant molecules within selected cells of a heterogeneous population.
The instruments and techniques for this work are described along with an overview
of the fundamental principles behind this methodology.
The model organism studied in this work was the filamentous fungi, Neurospora
crassa, the orange bread mould. It is the best characterised of all the filamentous
fungi, a group of organisms that are critically important to agriculture, medicine and
the environment. Capillary electrophoresis electrospray mass spectrometry (CE-ESIMS)
was used to measure the intra-cellular concentration of disaccharides, in
particular trehalose. In Neurospora crassa this molecule is synthesised in response
to environmental stress, and has been reported to accumulate at concentrations as
high as 10 mM, based on measurements using bulk cell populations. The value of
1.3 mM for the intra-cellular concentration of disaccharide measured in the single
cell sampling experiments described in this thesis is in good agreement with this
previously published maximum concentration.
Following topical application of a commercially relevant fungicide, azoxystrobin, to
cell cultures of Neurospora crassa, the intra-cellular concentration of the fungicide
was measured. For cells treated with azoxystrobin at a concentration of 14.8 pM (the
saturation concentration of azoxystrobin in water), the intra-cellular concentration
was shown to reach 9.9 μM within 5 minutes. It is likely that the high surface to
volume ratio of the fungal hyphae facilitats the rapid diffusion of these large
hydrophobic molecules across the cell membrane.
The development of novel instrumentation applicable to the analysis of ultra-low
volume samples is presented, encompassing microsampling, transfer, ionisation and
detection. Their utility in comparison with competing techniques is discussed, along
with suggestions as to the expected development of this technique and possible
directions for future work
Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls
Background and aims The softening of ripening fruit involves partial depolymerisation of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of homogalacturonan and rhamnogalacturonan-I (RG-I) domains of pectin respectively. However, the important biological question of whether RGL exhibits action in vivo had not been tested.Methods We developed a method for specifically and sensitively detecting in-vivo RGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated ‘fingerprint’ product (tetrasaccharide) of RGL action. Key Results In model experiments, potato RG-I that had been partially cleaved in vitro by commercial RGL was digested by Driselase, releasing an unsaturated tetrasaccharide (‘UA-Rha-GalA-Rha’), taken as diagnostic of RGL action. This highly acidic fingerprint compound was separated from monosaccharides (galacturonate, galactose, rhamnose etc.) by electrophoresis at pH 2, then separated from UA–GalA (the fingerprint of pectate lyase action) by thin-layer chromatography (TLC). The ‘UA-Rha-GalA-Rha’ was confirmed as 4-deoxy--L-threo-hex-4-enopyranuronosyl-(12)-L-rhamnosyl-(14)-D-galacturonosyl-(12)-L-rhamnose by mass spectrometry and acid hydrolysis. Driselase digestion of cell walls from diverse ripe fruits [date, sea buckthorn, cranberry, yew (arils), mango, plum, blackberry, apple, pear and strawberry] yielded the same fingerprint compound, demonstrating that RGL had been acting in vivo in these fruits prior to harvest. The ‘fingerprint’ : (galacturonate + rhamnose) ratio in digests from ripe dates was approximately 1:72 (mol/mol), indicating that ~1.4% of the backbone RhaGalA bonds in endogenous RG-I had been cleaved by in-vivo RGL action. Conclusions The results provide the first demonstration that RGL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening. <br/
Metabolites of 2,3-diketogulonate delay peroxidase action and induce non-enzymic H2O2 generation : Potential roles in the plant cell wall
A proportion of the plant's L-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolitesmay act as pro-oxidants and anti-oxidants. One ascorbate metabolite is 2,3-dilcetogulonate (DKG), preparations of which can non-enzymically generate H2O2 and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate H2O2 and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with lambda(max) 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major H2O2-generating and peroxidase-delaying principle. Compound (1) was slowly destroyed by ascorbate oxidase, and was less stable at pH 6 than at pH 1. Electrophoresis of an HPLC-enriched preparation of (1) suggested a strongly acidic (pK(a) approximate to 2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula C6H6O5, i.e. it is a reduction product of DKG (C6H8O7). In conclusion, compound (1) is the major H2O2-generating, peroxidase-delaying principle formed non-enzymically from DKG in the pathway ascorbate -> dehydroascorbic acid -> DKG -> (1). We hypothesise that (1) generates apoplastic H2O2 (and consequently hydroxyl radicals) and delays cell-wall crosslinking - both these effects favouring wall loosening, and possibly playing a role in pathogen defence. (C) 2017 The Authors. Published by Elsevier Inc.Peer reviewe
Complementary Ionization Techniques for the Analysis of Scotch Whisky by High Resolution Mass Spectrometry
Fourier
transform mass spectrometry (FTMS) is widely used to characterize
the chemical complexity of mixtures, such as natural organic matter
(NOM), petroleum, and agri-food products (including Scotch whisky).
Although electrospray ionization (ESI) is by far the most widely used
ionization source in these studies, other ionization techniques are
available and may offer complementary information. In a recent study,
we found matrix free laser desorption/ionization (LDI) to be effective
for the analysis of Suwannee river fulvic acid (SRFA), and to provide
complementary chemical insights. In this study, LDI along with atmospheric
pressure photoionization (APPI) and atmospheric pressure chemical
ionization (APCI) were compared to ESI for the analysis of Scotch
whisky. High mass accuracy (54 ppb, mean) allowed for the assignment
of 86% of peaks, with 3993 unique molecular formulas identified from
four representative samples analyzed. All four ionization techniques,
performed in negative mode, identified thousands of formulas. Many
were unique to each ionization source, while 699 formulas were common
to all techniques. Ions were identified in both deprotonated and radical
anion forms. Our study highlights the importance of a multi-ionization
source approach; we recommend that analysis of complex mixtures, especially
novel ones, should not be limited solely to ESI
An optimised small-scale sample preparation workflow for historical dye analysis using UHPLC-PDA applied to Scottish and English Renaissance embroidery
A sample preparation workflow for historical dye analysis based on 96 well plates and filtration by centrifugation was developed. It requires less sample and the introduced error is decreased, making it useful for culturally important objects.
A sample preparation workflow for historical dye analysis requiring less sample has been developed. Samples as small as 0.01 ± 0.005 mg have been successfully analysed and high percentage recoveries (>85%), more automation and shorter preparation time have been achieved using filtration by centrifugation and only one manual transfer. The optimised workflow based on 96 well plates together with the shorter UHPLC method developed makes dye analysis data collection faster from unprocessed sample to result, facilitating the creation of larger datasets and application of chemometric approaches. The method was evaluated on 85 samples from 12 dye sources (RSD < 5.1%, = 5) as well as 22 samples from a 17 century embroidered stomacher from the National Museums Scotland (NMS) collection
Spatial Localization of Vitamin D Metabolites in Mouse Kidney by Mass Spectrometry Imaging
Vitamin D plays a key role in the maintenance of calcium/phosphate homeostasis and elicits biological effects that are relevant to immune function and metabolism. It is predominantly formed through UV exposure in the skin by conversion of 7-dehydrocholsterol (vitamin D3). The clinical biomarker, 25-hydroxyvitamin D (25-(OH)-D), is enzymatically generated in the liver with the active hormone 1,25-dihydroxyvitamin D then formed under classical endocrine control in the kidney. Vitamin D metabolites are measured in biomatrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In LC-MS/MS, chemical derivatization (CD) approaches have been employed to achieve the desired limit of quantitation. Recently, matrix-assisted laser desorption/ionization (MALDI) has also been reported as an alternative method. However, these quantitative approaches do not offer any spatial information. Mass spectrometry imaging (MSI) has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. On-tissue chemical derivatization (OTCD) enables MSI to image molecules with poor ionization efficiently. In this technical report, several derivatization reagents and OTCD methods were evaluated using different MSI ionization techniques. Here, a method for detection and spatial distribution of vitamin D metabolites in murine kidney tissue sections using an OTCD-MALDI-MSI platform is presented. Moreover, the suitability of using the Bruker ImagePrep for OTCD-based platforms has been demonstrated. Importantly, this method opens the door for expanding the range of other poor ionizable molecules that can be studied by OTCD-MSI by adapting existing CD methods
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