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No / Lipidomics aim to generate qualitative and quantitative information on different classes of lipids and their species, and when applied in conjunction with proteomic and genomic assays, facilitate the comprehensive study of lipid metabolism in cellular, organ or body systems. Advances in mass spectrometry have underpinned the expansion of lipidomic methodologies. Prostanoids are potent autacoids present in a plethora of cellular systems, known best for their intimate role in inflammation. Electrospray ionisation (ESI) allows the efficient ionisation of prostanoids in aqueous systems. ESI can be readily coupled to liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS)-based detection, thus allowing the development of a potent and selective LC/ESI-MS/MS quantitative assays. The protocol we describe in this chapter outlines the steps we follow to a) extract prostanoids from solid or liquid samples, b) semi-purify the metabolites using solid phase extraction c) set-up the HPLC separation using reverse phase chromatography and d) set up the MS/MS assay using a triple quadrupole mass spectrometer. The experimental details and notes presented here are based on the detailed protocols followed in our group

Yes / Sunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B-induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids and their subsequent metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and for 72h examined (i) expression of pro- and anti-inflammatory eicosanoids using LC/ESI-MS/MS and (ii) immunohistochemical expression of COX-2, 12-LOX, 15-LOX and leucocyte markers, while (iii) quantifying clinical erythema. We show that vasodilatory prostaglandins (PG)E2, PGF2¿ and PGE3 accompany the erythema in the first 24-48h, associated with increased COX-2 expression at 24h. Novel, potent leukocyte chemoattractants 11-, 12- and 8-monohydroxy-eicosatetraenoic acid (-HETE) are elevated from 4-72h, in association with peak dermal neutrophil influx at 24h, and increased dermal CD3+ lymphocytes and 12- and 15-LOX expression from 24-72h. Anti-inflammatory metabolite 15-HETE shows later expression, peaking at 72h. Sunburn is characterized by overlapping phases of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution. / The Wellcome Trust

Yes / The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including ‘palaeoshellomics’, the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of ‘double-buttons’. This craft is found throughout Europe between 4200–3800 BCE, highlighting the ornament-makers’ profound knowledge of the biogeosphere and the existence of cross-cultural traditions. / Ministry of Education, Universities and Research Young Researcher: European Commission PERG-GA-2010-26842: Leverhulme Trust: Centre National de la Recherche Scientifique: Campus France, Universita` Italo-Francese PHC Galile´ programme

Pre-treatment with polyunsaturated fatty acids or bioactive lipid mediators has been shown to reduce neuronal injury in rodent models of focal ischaemia, but the molecular mechanisms underlying this neuroprotection are unclear. In this study, we aimed to investigate whether systemic administration of alpha linolenic acid (ALA) leads to changes in the profile of mouse brain phospholipid and bioactive lipid mediators in both mouse brain and plasma within the previously determined neuroprotection time window. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) allowed us to detect and identify 47 phospholipids in mouse cerebral cortex, including several phospholipid species not previously reported in brain lipidomic studies. These included a phosphatidylethanolamine species with m/z 720 that has been associated with retinal stem cells. No widespread changes in cerebral cortex phospholipid composition were observed following intravenous ALA. Several significant changes in lipid mediators (P<0.05 with two-way ANOVA and post hoc Dunnett's t test) were detected in ALA-treated animals compared to untreated and vehicle-injected animals. Many of the affected lipid mediators are ligands for prostanoid receptors which have been demonstrated to play a role in the development of brain injury following cerebral ischaemia, implying that changes in bioactive lipid mediators or modulation of prostanoid receptors may occur following ALA pre-treatment in mice. This study illustrates the potential of advanced lipidomic analysis as a novel tool for neurochemists.

Metabólitos secundários produzidos por actinobactérias são uma inesgotável fonte de compostos com potentes atividades biológicas e estruturas intrínsecas. O desenvolvimento em instrumentação analítica tem contribuído significantemente para acelerar o processo de identificação e caracterização desses metabólitos bioativos. Sem dúvida alguma, a espectrometria de massas (MS) e o seu acoplamento com técnicas de separação, especialmente a cromatografia líquida (UHPLC-MS), tem sido reconhecida como a técnica mais eficiente em análises de produtos naturais. Nesta dissertação foi explorado o potencial da espectrometria de massas como ferramenta analítica para a identificação e caracterização estrutural de fitotoxinas produzidas por actinobactérias isoladas da rizosfera de plantas da caatinga. Foram produzidos noventa extratos de actinobactérias, dos quais quinze apresentaram alguma atividade para o bioensaio da Lemna minor e seis apresentaram atividade para o bioensaio da Chlorella vulgaris. Os extratos brutos ativos das actinobactérias Caat 7-38, Caat 8-6 e Caat 5-29 foram selecionados para caracterização dos compostos ativos, os quais foram isolados empregando o fracionamento guiado por bioensaios. No extrato bruto Caat 7-38, a actinomicina D foi identificada como fitotoxina, ao passo que para o extrato bruto Caat 8-6, foi possível inferir a atividade fitotóxica à presença do griseorhodin A. Já para o extrato bruto Caat 5-29, o composto identificado com atividade fitotóxica apresenta uma estrutura inédita, provavelmente pertencente à classe das anguciclinonas. Foi realizado ainda um estudo para avaliar o efeito da adição de terras raras ao meio de cultivo da actinobacteria Caat 7-38. Para os meios de cultivos contendo neodímio e, principalmente, lantânio ocorreu uma superprodução da actinomicina D, indicando assim, o grande potencial da aplicação das terras raras nos estudos de micro-organismos. / Secondary metabolites produced by actinomycetes are an inexhaustible source of compounds with potent biological activities and intrinsic structures. The development analytical instrumentation has contributed significantly to accelerate the identification and characterization of these bioactive metabolites. Undoubtedly, mass spectrometry (MS) and its coupling with separation techniques, especially liquid chromatography (UHPLC-MS) has been recognized as the most \"efficient\" technique in natural product analysis. In this work was explored the potential of mass spectrometry as an analytical tool for identification and structural characterization of phytotoxins produced by actinomycetes isolated from the rhizosphere of plants from the Caatinga biome. Ninety actinomycetes extracts were produced, of which fifteen showed some activity for the bioassay with Lemna minor and six showed activity for the bioassay with Chlorella vulgaris. The crude active extract of actinomycetes Caat 7-38, Caat 8-6 and Caat 5-29 were selected to characterize the active compounds, which were isolated using bioassay-guided fractionation. In the crude extract Caat 7-38, actinomycin D was identified as phytotoxin, while for crude extract Caat 8-6, it was possible to infer phytotoxic activity to the presence of griseorhodin A. For the crude extract Caat 5-29, the compound identified with phytotoxic activity presents a new structure, probably belonging to the class of anguciclinones. A study to evaluate the effect of addition of rare earths to the culture medium of actinobacteria Caat 7-38 was also carried out. To the culture medium containing neodymium and especially lanthanum occurred overproduction of actinomycin D, thus indicating the great potential of application of rare earths in the studies of microorganisms.

Mass spectrometry is a versatile analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry further expands the applications of mass spectrometry, making it more than a mere detector. With tandem mass spectrometry, the mass spectrometer is capable of probing reaction mechanisms, monitoring reaction processes, and performing fast analysis on complex samples. In tandem mass spectrometry, after activation the precursor ions fragment into small fragment ions through one or more pathways, which are affected by the ion’s inherit property, the ion type, and the activation method. To obtain complementary information, one can alter the fragmentation pathway by changing the ion via ion charge manipulation and covalent modification to the ion. Gas-phase ion/ion reactions provide an easy approach to changing ion type and facile modification to the analyte ions. It has been extensively used for spectrum simplification and analyte structural studies. In this dissertation, ion/ion reaction facilitated mass spectrometry methods are studied, and explorations into the method development involving front-end mass spectrometer are discussed.<br>The first work demonstrates a special rearrangement reaction for gas-phase Schiff-base-modified peptides. Gas-phase Schiff-base modification of peptides has been applied to facilitate the primary structural characterization via tandem mass spectrometry. A major or minor fragment pathway related to the novel rearrangement reaction was observed upon in-trap collisional activation of the gas-phase Schiff-base-modified peptides. The rearrangement reaction involves the imine of the Schiff base and a nucleophile present in the polypeptide. The occurrence of the rearrangement reaction is affected by several factors, such as ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. The rearrangement reaction does not affect the amount of structural information that can be obtained by collisional activation of the Schiff-base-modified peptide, but when the rearrangement reaction is dominant, it can siphon away signal from the structurally diagnostic processes.<br>Efforts have also been put into the method development of peptide and protein aggregation detection via electrospray ionization mass spectrometry (ESI-MS). People have studied peptide and protein aggregation processes to understand the mechanism of amyloid-related diseases and to control the quality of the peptide and protein pharmaceuticals. ESI-MS is suitable for solution aggregation studies because of its compatibility with solution samples and the straightforward result of the analyte’s oligomeric state on the mass spectrum. However, peak overlap issue and nonspecific aggregation in the ESI process can obscure the result. Here, the application of proton transfer ion/ion reaction to the analyte has been found useful to reduce or eliminate the peak overlap issue. A statistical model based on Poisson statistics has been proposed to deal with the ESI-induced nonspecific aggregation in the droplet and to differentiate the solution-phase aggregation from the droplet-induced aggregation. Factors that affect the accuracy of the statistical model have been discussed with MATLAB simulations.<br>In the era of biological system studies, sample complexity is a challenge every analytical chemist has to face. The analysis of complex sample can be facilitated by the combination of separation techniques outside the mass spectrometer (such as differential mobility spectrometry (DMS)) and ion structure probing techniques inside the mass spectrometer (such as tandem mass spectrometry and gas-phase ion/ion reactions). Here the coupling method between DMS and ion/ion reaction is developed and tested with model peptide systems to demonstrate its possible application in complex sample characterization such as isomer identification.<br>

<div>Amongst the various classes of mass analyzers, the quadrupole ion trap (QIT) is by far the most versatile. Although it can achieve only modest resolution (unit) and mass accuracy (101-102 ppm), it has high sensitivity and selectivity, can operate at pressures exceeding 10-3 torr, is tolerant to various electrode imperfections, and has single analyzer tandem mass spectrometry (MS/MS) capabilities in the form of product ion scans. These characteristics make the QIT ideal for mass spectrometer miniaturization, as most of the fundamental performance metrics of the QIT do not depend on device size. As such, the current drive in miniature systems is to adopt miniature ion traps in various forms – 3D, linear, toroidal, rectilinear, cylindrical, arrays, etc.</div><div><br></div><div>Despite being one of the two common mass analyzers with inherent MS/MS capabilities (the other being the Fourier transform ion cyclotron resonance mass spectrometer), it is commonly accepted that the QIT cannot perform one-dimensional precursor ion scans and neutral loss scans - the other two main MS/MS scan modes - or two-dimensional MS/MS scans. The former two are usually conducted in triple quadrupole instruments in which a first and third quadrupole are used to mass select precursor and product ions while fragmentation occurs in an intermediate collision cell. The third scan can be accomplished by acquiring a product ion scan of every precursor ion, thus revealing the entire 2D MS/MS data domain (precursor ion m/z vs. product ion m/z). This, however, is not one scan but a set of scans. Because the ion trap is a tandem-in-time instrument rather than a tandem-in-space analyzer, precursor ion scans, neutral loss scans, and 2D MS/MS are, at best, difficult.</div><div><br></div><div>Yet miniature mass spectrometers utilizing quadrupole ion traps for mass analysis would perhaps benefit the most from precursor scans, neutral loss scans, and 2D MS/MS because they generally have acquisition rates (# scans/s) an order of magnitude lower than their benchtop counterparts. This is because they usually use a discontinuous atmospheric pressure interface (DAPI) to reduce the gas load on the backing pumps, resulting in a ~1 scan/s acquisition rate and making the commonly-used data-dependent acquisition method (i.e. obtaining a product ion scan for every abundant precursor ion) inefficient in terms of sample consumption, time, and instrument power. Precursor and neutral loss scans targeting specific molecular functionality of interest - as well as 2D MS/MS – are more efficient ways of moving through the MS/MS data domain and thus pair quite readily with miniature ion traps.</div><div><br></div><div>Herein we demonstrate that precursor ion scans, neutral loss scans, and 2D MS/MS are all possible in a linear quadrupole ion trap operated in the orthogonal double resonance mode on both benchtop and portable mass spectrometers. Through application of multiple resonance frequencies matching the secular frequencies of precursor and/or product ions of interest, we show that precursor ions can be fragmented mass-selectively and product ions ejected simultaneously, preserving their relationship, precursor ion -> product ion + neutral, in the time domain and hence allowing the correlation between precursor and product ions without prior isolation. By fixing or scanning the resonance frequencies corresponding to the targeted precursor and product ions, a precursor ion scan or neutral loss scan can be conducted in a single mass analyzer. We further show that 2D MS/MS - acquisition of all precursor ion m/z values and a product ion mass spectrum for every precursor ion, all in a single scan - is possible using similar methodology. These scan modes are particularly valuable for origin-of-life and forensic applications for which the value of miniature mass spectrometers is readily evident.</div>

Protein identification using tandem mass spectrometry (MS/MS) has proven to be an effective way to identify proteins in a biological sample. An observed spectrum is constructed from the data produced by the tandem mass spectrometer. A protein can be identified if the observed spectrum aligns with the theoretical spectrum. However, data generated by the tandem mass spectrometer are affected by errors thus making protein identification challenging in the field of proteomics. Some of these errors include wrong calibration of the instrument, instrument distortion and noise. In this thesis, we present a pre-processing method, which focuses on the removal of noisy data with the hope of aiding in better identification of proteins. We employ the method of binning to reduce the number of noise peaks in the data without sacrificing the alignment of the observed spectrum with the theoretical spectrum. In some cases, the alignment of the two spectra improved.