165 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 ÎŒM 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
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
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 âfingerprintâ 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 (âUA-Rha-GalA-Rhaâ), 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 UAâGalA (the fingerprint of pectate lyase action) by thin-layer chromatography (TLC). The âUA-Rha-GalA-Rhaâ 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 âfingerprintâ : (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/
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
Native Ion Mobility Mass Spectrometry (IMâMS) reveals that small organic acid fragments impart gasâphase stability to carbonic anhydrase II
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
Fruit softening:Evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls
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Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins
Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433â434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down analysis of larger proteins (up to âŒ50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of âŒ3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experimentsâhighlighting advantages for top-down LCâMS/MS workflows and top-down proteomics
Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition
Contemporary medicinal chemistry considers fragmentâbased drug discovery (FBDD) and inhibition of proteinâprotein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nanoâESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Followâup biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 Câterminal domain. An inâsilico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit doseâdependent inhibitor of the target PPI
On-Tissue Chemical Derivatization in Mass Spectrometry Imaging
Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrixâassisted laser desorption/ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed. However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed onâtissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCDâMSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive
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