Scedosporium and Pseudallescheria low molecular weight metabolites revealed by database search

The potential of mMass software search tool with new compound libraries was demonstrated on metabolomics of Scedosporium prolificans, S. apiospermum and Pseudallescheria boydii sensu stricto. Cyclic peptides pseudacyclins, small molecular weight tyroscherin analogues and various lipids were annotated by open source mass spectometry tool utilising accurate matrix-assisted laser desorption/ionisation mass spectral data of intact fungal spores. Electrospray ionisation combined with tandem mass spectrometry was used for monohexosylceramide characterisation in fungal extracts

A proteomic approach for the rapid, multi-informative and reliable identification of blood

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence in the forensic investigation of serious offences. Because many of the current enhancement methods used by crime scene investigators are presumptive, the visualisation of blood is not always reliable nor does it bear additional information. In the work presented here, two methods employing a shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols employ both an in solution digestion method and a recently proposed procedure involving immobilization of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes). Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate

Development of operational protocols for the analysis of primary and secondary fingermark lifts by MALDI-MS Imaging

Eight years of intensive research have demonstrated that Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Profiling and Imaging (MALDI-MSI and MSP) are powerful tools to gather intelligence around a suspect lifestyle, directly from the identifying ridges of a latent fingermark. In the past three years, many efforts have been invested into translating laboratory methodologies to the field; this was undertaken by devising protocols either for (a) enabling initial fingermark visualisation, such as through the Dry-Wet method, recovery and subsequent MALDI MS based analysis, or for (b) rendering the MS methodologies compatible with the prior application of commonly employed fingermark enhancement techniques (FET). In the present work a major point of interest concerned the sample treatment of FET visualised-lifted fingermarks and the subsequent MS performance of primary tape lifted fingermarks ("primary lifts") versus secondary tape lifted fingermarks (recovery from the surface a second time following the initial primary lift). This was necessary since it may not always be possible to obtain primary lifts of marks visualised at crime scenes for remote MALDI-MSP and MSI. The work illustrated here has provided methodological insights into establishing how to best treat a few types of developed marks in preparation for MALDI-MSI when presented as both secondary and primary lifts; it was demonstrated, as expected, that primary lifts generally yield much higher quality chemical/physical information and are therefore crucial to maximise chances of suspect identification and of retrieval of chemical intelligence. When analysing secondary lifted marks that have been initially developed using aluminium or carbon powders, any of the trialled sample preparation methodologies can be employed except the Dry-Wet method. In the case of TiO2 powder developed marks, the best ridge coverage was achieved by re-enhancing the mark using the initial powder and spray-coating with MALDI matrix. Primary lifts of fingermarks contaminated with an exogenous substance (used as a reference model) yielded the best ridge detail quality whilst for secondary lifts of natural marks pre-enhanced with aluminium powder, significantly greater intensity of the ion image was observed for the sections subjected to either no further enhancement or re-enhancement using aluminium powder. Keywords: Fingermarks; MALDI; Imaging; powders; lift

Optimization of sample preparation and instrumental parameters for the rapid analysis of drugs of abuse in hair samples by MALDI-MS/MS imaging

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) has been employed to rapidly screen longitudinally sectioned drug user hair samples for cocaine and its metabolites using continuous raster imaging. Optimization of the spatial resolution and raster speed were performed on intact cocaine contaminated hair samples. The optimized settings (100 × 150 μm at 0.24 mm/s) were subsequently used to examine longitudinally sectioned drug user hair samples. The MALDI-MS/MS images showed the distribution of the most abundant cocaine product ion at m/z 182. Using the optimized settings, multiple hair samples obtained from two users were analyzed in approximately 3 h: six times faster than the standard spot-to-spot acquisition method. Quantitation was achieved using longitudinally sectioned control hair samples sprayed with a cocaine dilution series. A multiple reaction monitoring (MRM) experiment was also performed using the 'dynamic pixel' imaging method to screen for cocaine and a range of its metabolites, in order to differentiate between contaminated hairs and drug users. Cocaine, benzoylecgonine, and cocaethylene were detectable, in agreement with analyses carried out using the standard LC-MS/MS method. Graphical Abstract ᅟ

Proteomics goes forensic: detection and mapping of blood signatures in fingermarks

A bottom up in situ proteomic method has been developed enabling the mapping of multiple blood signatures on the intact ridges of blood fingermarks byMatrix Assisted Laser Desorption Mass Spectrometry Imaging (MALDI-MSI). This method, at a proof of concept stage, builds upon recently published work demonstrating the opportunity to profile and identify multiple blood signatures in bloodstains via a bottom up proteomic approach. The present protocol addresses the limitation of the previously developed profiling method with respect to destructivity; destructivity should be avoided for evidence such as blood fingermarks, where the ridge detail must be preserved in order to provide the associative link between the biometric information and the events of bloodshed. Using a blood mark reference model, trypsin concentration and spraying conditions have been optimised within the technical constraints of the depositor eventually employed; the application of MALDI-MSI and Ion Mobility MS have enabled the detection, confirmation and visualisation of blood signatures directly onto the ridge pattern. These results are to be considered a first insight into a method eventually informing investigations (and judicial debates) of violent crimes in which the reliable and non-destructive detection and mapping of blood in fingermarks is paramount to reconstruct the events of bloodshed

The contribution of non-catalytic carbohydrate binding modules to the activity of lytic polysaccharide monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharidemonooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.This work was supported by the Spanish Ministerio de Ciencia e Innovación (grants BFU2009-07371 to A.C., BIO2009-10872 and BIO2010-15424 to A.M. and SAF2008-05742-C02-01 and CSD2008-00005 to J.L.N.) and the Generalitat Valenciana (grants ACOMP2006/083 and ACOMP2011/211 to A.C., ACOMP2010/114 and ACOMP2011/113 to J.L.N.). M.L. López-Redondo was a fellow of the Fundación Mutua Madrileña Automovilística

Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis

Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs

The analysis of latent fingermarks on polymer banknotes using MALDI-MS

In September 2016, the UK adopted a new Bank of England (BoE) £5 polymer banknote, followed by the £10 polymer banknote in September 2017. They are designed to be cleaner, stronger and have increased counterfeit resilience; however, fingermark development can be problematic from the polymer material as various security features and coloured/textured areas have been found to alter the effectiveness of conventional fingermark enhancement techniques (FETs). As fingermarks are one of the most widely used forms of identification in forensic cases, it is important that maximum ridge detail be obtained in order to allow for comparison. This research explores the use of matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) profiling and imaging for the analysis of fingermarks deposited on polymer banknotes. The proposed methodology was able to obtain both physical and chemical information from fingermarks deposited in a range of scenarios including; different note areas, depletion series, aged samples and following conventional FETs. The analysis of forensically important molecular targets within these fingermarks was also explored, focussing specifically on cocaine. The ability of MALDI-MS to provide ridge detail and chemical information highlights the forensic applicability of this technique and potential for the analysis of fingermarks deposited onto this problematic surface