Top-down shotgun lipidomics relies on direct infusion of total lipid extracts into a high-resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top-down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high-resolution spectra in both polarity modes with sub-ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within 7 min acquisition cycle.

Abstract. Here we report on the application of a novel shotgun lipidomics platform featuring an Orbitrap Fusion mass spectrometer equipped with an automated nanoelectrospray ion source. To assess the performance of the platform for indepth lipidome analysis, we evaluated various instrument parameters, including its high resolution power unsurpassed by any other contemporary Orbitrap instrumentation, its dynamic quantification range and its efficacy for in-depth structural characterization of molecular lipid species by quadrupole-based higher-energy collisional dissociation (HCD), and ion trap-based resonant-excitation collision-induced dissociation (CID). This evaluation demonstrated that FTMS analysis with a resolution setting of 450,000 allows distinguishing isotopes from different lipid species and features a linear dynamic quantification range of at least four orders of magnitude. Evaluation of fragmentation analysis demonstrated that combined use of HCD and CID yields complementary fragment ions of molecular lipid species. To support global lipidome analysis, we designed a method, termed MS ALL , featuring high resolution FTMS analysis for lipid quantification, and FTMS 2 analysis using both HCD and CID and ITMS 3 analysis utilizing dual CID for in-depth structural characterization of molecular glycerophospholipid species. The performance of the MS ALL method was benchmarked in a comparative analysis of mouse cerebellum and hippocampus. This analysis demonstrated extensive lipidome quantification covering 311 lipid species encompassing 20 lipid classes, and identification of 202 distinct molecular glycerophospholipid species when applying a novel high confidence filtering strategy. The work presented here validates the performance of the Orbitrap Fusion mass spectrometer for in-depth lipidome analysis.

Here we describe a novel surface sampling technique termed pressurized liquid extraction surface analysis (PLESA), which in combination with a dedicated high-resolution shotgun lipidomics routine enables both quantification and in-depth structural characterization of molecular lipid species extracted directly from tissue sections. PLESA uses a sealed and pressurized sampling probe that enables the use of chloroform-containing extraction solvents for efficient in situ lipid microextraction with a spatial resolution of 400 μm. Quantification of lipid species is achieved by the inclusion of internal lipid standards in the extraction solvent. The analysis of lipid microextracts by nanoelectrospray ionization provides long-lasting ion spray which in conjunction with a hybrid ion trap-orbitrap mass spectrometer enables identification and quantification of molecular lipid species using a method with successive polarity shifting, high-resolution Fourier transform mass spectrometry (FTMS), and fragmentation analysis. We benchmarked the performance of the PLESA approach for in-depth lipidome analysis by comparing it to conventional lipid extraction of excised tissue homogenates and by mapping the spatial distribution and molar abundance of 170 molecular lipid species across different anatomical mouse brain regions.

After just simple degassing, dilution, pH adjustment and direct flow injection, characteristic fingerprint spectra of beer samples have been obtained by fast (few seconds) electrospray ionization mass spectrometry (ESI-MS) analysis in both the negative and positive ion modes. A total of 29 samples belonging to the two main beer types (lagers and ales) and several beer subtypes from USA, Europe and Brazil could be clearly divided into three groups both by simple visual inspection of their ESI(+)-MS and ESI(-)-MS fingerprints as well as by chemometric treatment of the MS data. Diagnostic ions with contrasting relative abundances in both the positive and negative ion modes allow classification of beers into three major types: P = pale (light) colored (pilsener, pale ale), D = dark colored (bock, stout, porter, mild ale) and M = malt beer. For M beers, samples of a dark and artificially sweetened caramel beer produced in Brazil and known as Malzbiers were used. ESI-MS/MS on these diagnostic beer cations and anions, most of which are characterized as arising from ionization of simple sugars, oligosaccharides, and iso-alpha-acids, yield characteristic tandem mass spectra adding a second and optional MS dimension for improved selectivity for beer characterization by fingerprinting. Direct ESI-MS or ESI-MS/MS analysis can therefore provide fast and reliable fingerprinting characterization of beers, distinguishing between types with different chemical compositions. Other unusual polar components, impurities or additives, as well as fermentation defects or degradation products, could eventually be detected, making the technique promising for beer quality control.

The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is

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ABSTRACT:This article describes the combination of whole-body autoradiography with liquid extraction surface analysis (LESA) and mass spectrometry (MS) to study the distribution of the tachykinin neurokinin-1 antagonist figopitant and its metabolites in tissue sections of rats after intravenous administration of 5.0 mg/kg figopitant. An overview of autoradiography results is presented together with mass spectrometry identification and semiquantification of parent drug and its metabolites based on LESA-MS. The quality and accuracy of data generated by LESA-MS were assessed in comparison with classic tissue extraction, sample cleanup, and high-performance liquid chromatography analysis. The parent drug and the N-dealkylated metabolite M474(1) (BIIF 1148) in varying ratios were the predominant compounds in all tissues investigated. In addition, several metabolites formed by oxygenation, dealkylation, and a combination of oxygenation and dealkylation were identified. In summary, the LESA-MS technique was shown to be a powerful tool for identification and semiquantification of figopitant and its metabolites in different tissues and was complementary to quantitative whole-body autoradiography for studying the distribution. IntroductionQuantitative whole-body autoradiography (QWBA) is the imaging method of choice to investigate the distribution of drug-related radioactivity in all organs and tissues of an intact organism, such as an animal carcass. With this technique, information on the concentration of the entire drug-related radioactivity is gained. However, the actual molecular entity-parent compound or metabolites-and their respective proportions of the radioactivity remains unknown. The conventional approach to identify and quantify parent drug and metabolites in tissues is the preparation of organ homogenates and sample analysis by liquid chromatography combined with radiodetection and tandem mass spectrometry (MS/MS). However, this approach is labor-intensive, and some tissues (e.g., salivary glands) and tissue substructures (e.g., renal inner and outer medulla) can be difficult or even impossible to sample and extract. Surface sampling methods such as desorption electrospray ionization, laser desorption ionization, direct analyses in real time, or matrix-assisted laser desorption ionization can speed up analysis time and cut overall costs compared with classic tissue extraction and enable spatial resolution of different anatomical substructures within a given organ (Reyzer et al., 2003).Recently, a fully automated liquid extraction-based surface sampling method for mass spectrometry (MS) analyses of drugs and metabolites in thin tissue sections has been described (Van Berkel and Kertesz, 2009;Kertesz and Van Berkel, 2010). This liquid extraction surface analysis (LESA) method uses a liquid microjunction probe to extract the analytes directly from the surface followed by automated nano-electrospray analysis. Direct surface sampling methods deal with high sample complexity, because no chromatographic separation ...