Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Electrospray ionization mass spectrometry (ESI-MS) was utilized for the structural determination and quantitative analysis of individual phospholipid molecular species from subpicomole amounts of human erythrocyte plasma membrane phospholipids. The sensitivity of ESI-MS was 2-3 orders of magnitude greater than that achievable with fast-atom bombardment mass spectrometry (FAB-MS). Phospholipid structure determination and quantitative analysis with ESI-MS can be performed directly from chloroform extracts of biologic samples, obviating the need for prior chromatographic separation of phospholipid classes which has been necessary in FAB-MS phospholipid analyses. Furthermore, ESI-MS is uncomplicated by differential fragmentation of molecular ions and idiosyncratic surface desorption, allowing the quantitation of phospholipids with coefficients of determination (r2) > 0.99 and accuracies > 95%. More than 50 human erythrocyte plasma membrane phospholipid constituents were identified by direct ESI-MS analysis of chloroform extracts of plasma membranes derived from the equivalent of < 1 microliter of whole blood. The major ethanolamine glycerophospholipid subclass in erythrocyte plasma membranes was plasmenylethanolamine that was highly enriched in polyunsaturated fatty acids at the sn-2 position. Collectively, these results demonstrate that ESI-MS of phospholipids is an enabling strategy for the direct structural determination and quantitative analysis of subpicomole amounts of phospholipids from biologic samples. <s> BIB001 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research. <s> BIB002 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Electrospray ionization-mass spectrometry (ESI- MS) is a very promising tool for the analysis of phospholipid compositions, but is hampered by the fact that not all molecu- lar species are detected with equal efficiency. We studied this and other issues that need to be taken into account to obtain truly quantitative compositional data. The key findings were as follows: First, the instrument response for both saturated and unsaturated phospholipid species decreased with increas- ing acyl chain length. This effect became increasingly promi- nent with increasing overall lipid concentration. Second, the degree of acyl chain unsaturation also had a significant effect on instrument response. At the highest concentration studied (10 pmol/ m l), polyunsaturated species gave 40% higher inten- sity than the fully saturated ones. The effect of unsaturation diminished and nearly disappeared with progressive dilution. Third, the instrument response for the different head group classes varied markedly depending on the infusion solvent used. Notably, inclusion of ammonia in the infusion sol- vent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the spec- tra. The fact that instrument response is dependent on many structural features, overall lipid concentration, solvent com- position, and instrument settings makes it necessary to in- clude several internal standards for each phospholipid class to obtain accurate data. Preferably, both unsaturated and sat- urated standards should be used. Finally, we quantified the major phospholipid classes of BHK cells using ESI-MS. The data agreed closely with those obtained with thin-layer chro- matography and phosphorus analysis. This study indicates that quantitative compositional data can be obtained with ESI-MS, provided that proper attention is paid to experimen- tal details, particularly the choice of internal standards. — Koivusalo M., P. Haimi, L. Heikinheimo, R. Kostiainen, and P. Somerharju. Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsatur- ation, and lipid concentration on instrument response. J. Lipid Res. 2001. 42: 663-672. <s> BIB003 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Herein we describe a rapid, simple, and reliable method for the quantitative analysis and molecular species fingerprinting of triacylglycerides (TAG) directly from chloroform extracts of biological samples. Previous attempts at direct TAG quantitation by positive-ion electrospray ionization mass spectrometry (ESI/MS) were confounded by the presence of overlapping peaks from choline glycerophospholipids requiring chromatographic separation of lipid extracts prior to ESI/MS analyses. By exploiting the rapid loss of phosphocholine from choline glycerophospholipids, in conjunction with neutral-loss scanning for individual fatty acids, overlapping peaks in the ESI mass spectrum were deconvoluted generating a detailed molecular species fingerprint of individual TAG molecular species directly from chloroform extracts of biological samples. This method readily detects as little as 0.1 pmol of each TAG molecular species from chloroform extracts and is linear over a 1000-fold dynamic range. The sensitivity of individual TAG molecular species to ESI/MS/MS analyses correlated with the unsaturation index and inversely correlated with total aliphatic chain length of TAG. An algorithm was developed which identifies sensitivity factors, thereby allowing the rapid quantitation and molecular species fingerprinting of TAG molecular species directly from chloroform extracts of biological samples. <s> BIB004 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> A rapid, simple, and reliable method has been developed for the characterization and quantitation of ceramide molecular species directly from chloroform extracts of biological samples by electrospray ionization tandem mass spectrometry (ESI/MS/MS). By exploiting the differential fragmentation patterns of deprotonated ceramide ions, individual 2-hydroxy and nonhydroxy ceramide molecular species were readily identified by ESI/MS/MS with the neutral loss of fragments of mass 256.2 and 327.3 which correspond to sphingosine derivatives. The ions generated from the neutral loss of 256.2 (i.e., [M - H - 256.2](-)) are unique for ceramides with N-acyl sphingosine with the 18-carbon homolog. However, the sensitivity for nonhydroxy ceramides in ESI/MS/MS with the neutral loss of 256.2 is approximately threefold higher than that for 2-hydroxy ceramides. The ions resulting from the neutral loss of 327.3 (i.e., [M - H - 327.3](-)) are specific for 2-hydroxy ceramides. Additionally, all ceramides including both 2-hydroxy and nonhydroxy forms can be confirmed and accurately quantitated by ESI/MS/MS with the neutral loss of 240.2 after correction for (13)C isotope factors. This methodology demonstrated a 1000-fold linear dynamic range and a detection limit at the subfemtomole range and was applied to directly quantitate ceramide molecular species in chloroform extracts of biological samples including brain tissues and cell cultures. <s> BIB005 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> BACKGROUND ::: Lysophosphatidylcholine (LPC) has been suggested to play a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors. Initial studies provided evidence for a potential use of LPC as diagnostic maker. However, existing methodologies are of limited value for a systematic evaluation of LPC species concentrations because of complicated, time-consuming procedures. We describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC quantification. ::: ::: ::: METHODS ::: Crude lipid extracts of EDTA-plasma samples were used for direct flow injection analysis. LPC 13:0 and LPC 19:0 were added as internal standards, and the ESI-MS/MS was operated in the parent-scan mode for m/z 184. Quantification was achieved by standard addition. Data processing was highly automated by use of the mass spectrometer software and self-programmed Excel macros. ::: ::: ::: RESULTS ::: The calibrators LPC 16:0, LPC 18:0, and LPC 22:0 showed a linear response independent of sample dilution and plasma cholesterol concentration for both internal standards. The within-run imprecision (CV) was 3% for the major and 12% for the minor species, whereas the total imprecision was approximately 12% for the major and 25% for the minor species. The detection limit was <1 micromol/L. ::: ::: ::: CONCLUSION ::: The developed ESI-MS/MS methodology with an analysis time of 2 min/sample, simple sample preparation, and automated data analysis allows high-throughput quantification of distinct LPC species from plasma samples, which could be a valuable tool for the evaluation of LPC as diagnostic marker. <s> BIB006 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Cellular lipids are composed of linear combinations of aliphatic chains covalently attached to backbones and/or head groups that collectively represent the cellular lipidome. Herein we present a novel two-dimensional electrospray ionization mass spectrometric approach to fingerprint most of the major and many of the minor lipid classes in the hepatic cellular lipidome, which collectively represent >80% of the total lipid mass, directly from their chloroform extracts. Through lipid class-selective intrasource ionization and subsequent analysis of two-dimensional cross-peak intensities, the chemical identity and mass composition of individual molecular species of most mouse hepatic lipid classes were determined from its chloroform extract. This new integrated platform provides a robust foundation for the automated analysis of multidimensional mass spectra to advance the level of analytical scripting in lipidomics to a new plateau. <s> BIB007 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> The choline head group containing phosphatidylcholine (PC) and sphingomyelin (SPM) are major eukaryotic lipid components playing an important role in forming membrane microdomains and serve as precursor of signaling molecules. Both lipids can be monitored by positive ion mode electrospray tandem mass spectrometry using a parent ion scan of m/z 184. Although PC species appear at even m/z and SPM species at odd m/z, there may be a significant overlap of their isotopes. In order to separate PC and SPM species, an isotope correction algorithm was established, which utilizes calculated isotope percentages to correct the measured peak intensities for their isotopic overlap. We could demonstrate that this approach was applicable to correct the isotope overlap resulting from spiked PC and SPM species. Quantification was achieved by addition of different PC and SPM species prior to lipid extraction. The developed assay showed a precision, detection limit and robustness sufficient for routine analysis. Furthermore, an analysis time of only 1.3 min combined with automated data analysis using self-programmed Excel Macros allows high-throughput analysis. In summary, this assay may be a valuable tool for detailed lipid analysis of PC and SPM species in a variety of sample materials. <s> BIB008 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics. <s> BIB009 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D(7)-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform. <s> BIB010 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Kainate is a glutamate analog that has been widely used in pharmacological studies of neuronal injury related to ischemic conditions and epilepsy. While altered lipid metabolism has been implicated in kainate action, no study has yet investigated the associated changes in lipid metabolites on a systems scale. Here we describe a mass spectrometry-based approach for profiling of lipid mixtures in a nontargeted fashion. Combined with tandem mass spectrometry, this method aims to identify lipids that are altered between two conditions, the kainate-treated and the control hippocampal tissues. In addition to reductions in major phospholipids with mainly polyunsaturated fatty acyl chains, we find elevated levels of ions that correspond to acylated forms of phosphatidylethanolamines and ceramides. Acylated phosphatidylethanolamines are neuroprotective lipids and precursors for anandamide, which signals via cannabinoid receptors. Quantitative analysis of ceramides shows that many molecular species with different a... <s> BIB011 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Data-dependent acquisition of MS/MS spectra from lipid precursors enables to emulate the simultaneous acquisition of an unlimited number of precursor and neutral loss scans in a single analysis. This approach takes full advantage of rich fragment patterns in tandem mass spectra of lipids and enables their profiling by complex (Boolean) scans, in which masses of several fragment ions are considered within a single logical framework. No separation of lipids is required, and the accuracy of identification and quantification is not compromised, compared to conventional precursor and neutral loss scanning. <s> BIB012 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> We report a method for the identification and quantification of glycerophospholipid molecular species that is based on the simultaneous automated acquisition and processing of 41 precursor ion spectra, specific for acyl anions of common fatty acids moieties and several lipid class-specific fragment ions. Absolute quantification of identified species was linear within a concentration range of 10 nM-100 microM and was achieved by spiking into total lipid extracts a set of synthetic lipid standards with diheptadecanoyl (17:0/17:0) fatty acid moieties, representing six common classes of glycerophospholipids. The automated analysis of total lipid extracts was powered by a robotic nanoflow ion source and produced currently the most detailed description of the glycerophospholipidome. <s> BIB013 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Direct profiling of total lipid extracts on a hybrid LTQ Orbitrap mass spectrometer by high-resolution survey spectra clusters species of 11 major lipid classes into 7 groups, which are distinguished by their sum compositions and could be identified by accurately determined masses. Rapid acquisition of survey spectra was employed as a "top-down" screening tool that, together with the computational method of principal component analysis, revealed pronounced perturbations in the abundance of lipid precursors within the entire series of experiments. Altered lipid precursors were subsequently identified either by accurately determined masses or by in-depth MS/MS characterization that was performed on the same instrument. Hence, the sensitivity, throughput and robustness of lipidomics screens were improved without compromising the accuracy and specificity of molecular species identification. The top-down lipidomics strategy lends itself for high-throughput screens complementing ongoing functional genomics efforts. <s> BIB014 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for approximately 1 min per sample. <s> BIB015 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Herein, a systematic study on the identification and quantitation of choline-containing phospholipid molecular species, including choline glycerophospholipid (PC), lysoPC, and sphingomyelin (SM), is described using multi-dimensional mass spectrometry-based shotgun lipidomics after intrasource separation (MDMS-SL). Current methods for analysis of choline-containing lipids were improved through multiple modifications leading to: (1) identification of constituents present in the PC and SM classes, subclasses of PC, and individual molecular species using MDMS-SL analysis in the positive-ion mode; (2) identification of the fatty acyl constituents and their regiospecificity in diacyl PC molecular species through the neutral loss of trimethylamine plus fatty acids; (3) direct identification of the alkenyl chains of plasmenylcholine species using precursor ion scans of the fragment ions carrying the alkenyl chains; (4) elimination of the effects of polyunsaturation on the quantitation of PC species by multiple ratiometric comparisons; (5) accurate identification and quantitation of lysoPC molecular species including regioisomers by diagnostic fragment ions; and (6) accurate identification and quantitation of SM molecular species by neutral loss scans of phosphocholine plus methyl aldehyde which is specific to SM molecular species. With these enhancements, the application of MDMS-SL for the analyses of choline-containing phospholipid molecular species in biomedical research has been extended to a much larger number of molecular species with greater quantitative accuracy and an increased depth of structural information. <s> BIB016 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. Aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. ::: Methods: Human serum lipoproteins VLDL, LDL and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 μl serum. Subsequently, phosphatidylcholine, lysophosphatidyl¬choline, sphingo-myelin, ceramide, phosphatidylethanolamine, phosphatidylethanolamine-based plasmalogen, cholesterol and cholesteryl ester content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). ::: ::: Results: Analysis of FPLC fractions with polyacrylamide gel electrophoresis demonstrated that albumin partially co-elutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant co-elution with the albumin containing fractions. About 60% of serum ceramide were found in LDL fractions and 60% of phosphatidylcholine, phosphatidylethanolamine and plasmalogens in HDL fractions. VLDL, LDL and HDL displayed characteristic lipid class and species pattern. ::: ::: Conclusion: The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort. <s> BIB017 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided approximately 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches. <s> BIB018 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes. <s> BIB019 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Technological advances in mass spectrometry and meticulous method development have produced several shotgun lipidomic approaches capable of characterizing lipid species by direct analysis of total lipid extracts. Shotgun lipidomics by hybrid quadrupole time-of-flight mass spectrometry allows the absolute quantification of hundreds of molecular glycerophospholipid species, glycerolipid species, sphingolipid species and sterol lipids. Future applications in clinical cohort studies demand detailed lipid molecule information and the application of high-throughput lipidomics platforms. In this review we describe a novel high-throughput shotgun lipidomic platform based on 96-well robot-assisted lipid extraction, automated sample infusion by mircofluidic-based nanoelectrospray ionization, and quantitative multiple precursor ion scanning analysis on a quadrupole time-of-flight mass spectrometer. Using this platform to compile comprehensive lipid arrays associated with metabolic dysfunctions is a powerful strategy for pinpointing the mechanistic details by which alterations in tissue-specific lipid metabolism are directly linked to the etiology of many lipid-mediated disorders. <s> BIB020 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> BACKGROUND ::: Dyslipoproteinemia, obesity and insulin resistance are integrative constituents of the metabolic syndrome and are major risk factors for hypertension. The objective of this study was to determine whether hypertension specifically affects the plasma lipidome independently and differently from the effects induced by obesity and insulin resistance. ::: ::: ::: METHODOLOGY/PRINCIPAL FINDINGS ::: We screened the plasma lipidome of 19 men with hypertension and 51 normotensive male controls by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer. The analysis encompassed 95 lipid species of 10 major lipid classes. Obesity resulted in generally higher lipid load in blood plasma, while the content of tri- and diacylglycerols increased dramatically. Insulin resistance, defined by HOMA-IR >3.5 and controlled for BMI, had little effect on the plasma lipidome. Importantly, we observed that in blood plasma of hypertensive individuals the overall content of ether lipids decreased. Ether phosphatidylcholines and ether phosphatidylethanolamines, that comprise arachidonic (20:4) and docosapentaenoic (22:5) fatty acid moieties, were specifically diminished. The content of free cholesterol also decreased, although conventional clinical lipid homeostasis indices remained unaffected. ::: ::: ::: CONCLUSIONS/SIGNIFICANCE ::: Top-down shotgun lipidomics demonstrated that hypertension is accompanied by specific reduction of the content of ether lipids and free cholesterol that occurred independently of lipidomic alterations induced by obesity and insulin resistance. These results may form the basis for novel preventive and dietary strategies alleviating the severity of hypertension. <s> BIB021 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet. <s> BIB022 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Higher energy collision dissociation (HCD) is a complementary fragmentation tool that has recently become available on mass spectrometers of the LTQ Orbitrap family. We report on a shotgun bottom-up lipidomics approach that relies on HCD of the isolated lipid precursors. HCD, together with the high mass resolution and mass accuracy of the Orbitrap analyzer, improved the confidence of molecular species assignment and accuracy of their quantification in total lipid extracts. These capabilities were particularly important for accounting for biologically interesting lipid species comprising polyunsaturated and odd numbered fatty acid moieties. We argue that now both bottom-up and top-down shotgun lipidomics could be performed on the same instrumentation platform. <s> BIB023 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Direct Infusion <s> Since our last comprehensive review on multi-dimensional mass spectrometry-based shotgun lipidomics (Mass Spectrom. Rev. 24 (2005), 367), many new developments in the field of lipidomics have occurred. These developments include new strategies and refinements for shotgun lipidomic approaches that use direct infusion, including novel fragmentation strategies, identification of multiple new informative dimensions for mass spectrometric interrogation, and the development of new bioinformatic approaches for enhanced identification and quantitation of the individual molecular constituents that comprise each cell's lipidome. Concurrently, advances in liquid chromatography-based platforms and novel strategies for quantitative matrix-assisted laser desorption/ionization mass spectrometry for lipidomic analyses have been developed. Through the synergistic use of this repertoire of new mass spectrometric approaches, the power and scope of lipidomics has been greatly expanded to accelerate progress toward the comprehensive understanding of the pleiotropic roles of lipids in biological systems. <s> BIB024
The first ones to propose lipidomic analysis by ESI were Han and Gross in 1994 BIB001 . This concept utilized the emerging combination of ESI with a triple quadrupole analyzer. Sample injection was done by a syringe pump. A few years later, the same instrumentation was successfully used for lipid analysis in combination with the first prototypes of a static nano ESI source  without syringe pump. The nano ESI source with a flow rate of about 80 nL/min resulted in higher ionization efficiencies than a syringe pump running at several µL/min. Although this method now widely termed 'shotgun lipidomics' has improved a lot in the last 15 years, the basic concept behind it remains the same. It is based on precursor ion and constant neutral loss scans of readily ionizable phospholipid headgroups, resulting in lipid class specific fragments BIB024 . Shotgun lipidomics avoids difficulties with concentration alterations and chromatographic abnormalities. Another advantage compared to LC-MS is the longer time which can be spent on each lipid class specific scan type. Addition of one internal standard per lipid class was shown to be sufficient for quantitation BIB016 , because ionization of lipids is largely dependent on the class specific head group and not so much on the fatty acyl chains BIB005 . As there are also contradictory reports about the influence of fatty acyl chain length and unsaturation on ionization efficiency  BIB003 , it is advisable to extensively evaluate each individual system on this issue. One drawback of shotgun lipidomics are isobaric overlaps of the M + 2 isotope with the monoisotopic peak of the compound with one double bond less. This can be overcome by deisotoping algorithms BIB007 . The output format of data usually indicates the sum of fatty acyl carbons and the sum of double bonds, but not the individual composition of fatty acids. Multi-dimensional mass spectrometry based shotgun lipidomics (MDMS-SL) is a further development of shotgun lipidomics taking into account the concept of building blocks in lipid structures BIB009 . MDMS-SL takes advantage of differential intrasource separation properties with various additives like Li + , NH 4 + or Na + , and unique fragments for each lipid class. Glycerolipids without class specific fragments are detected by constant neutral losses of fatty acids and information about the intact lipid is drawn from the combinatorial possibilities of all monitored fatty acid neutral losses BIB004 . Coupled with the Nanomate® system (Advion Biosciences, Ithaca, NY), the MDMS-SL concept proves to be a powerful high throughput device because of its high degree of automatization and the enhanced sensitivity provided by a nano-ESI source. The MDMS-SL system covers quantitative analysis of various classes of glycerophospholipids, sphingolipids and glycerolipids BIB024 . Flow injection lipidomic analysis, a variation of shotgun lipidomics is proposed by the group of Liebisch BIB017 . In contrast to classical shotgun lipidomic methods, this experimental setup utilizes an HPLC apparatus coupled to a triple quadrupole analyzer. The HPLC pump runs at microflow rates and the autosampler injects samples automatically into the flow, which delivers the sample directly into the ESI source without HPLC column. The HPLC autosampler offers a higher degree of automatization than a syringe pump. Furthermore, it results in a short and concentrated sample pulse for about one minute, which can be used for data acquisition with precursor ion and constant neutral loss scans. Depending on the number of scans necessary, multiple injections per sample are possible. Quantitation is achieved by a standard addition method with multiple standard curves, featuring one internal standard and sets of lipids with different fatty acyl chain lengths and degrees of unsaturation BIB008 . The method is very robust, highly automated and was applied on various subclasses of glycerophospholipids, sphingolipids and sterols BIB008 BIB006 BIB002 BIB010 . As for all low resolution direct infusion technologies it runs into its limits when isobaric nominal mass compounds derived from the same phospholipid subclass occur, like, e.g., diacyl and acyl-alkyl glycerophospholipids. Another direct infusion approach encompasses coupling of a syringe pump and a triple quadrupole analyzer in multiple reaction monitoring (MRM) mode BIB011 . In this experimental setup, the anticipated precursor and product ions must be known. It allows a quick and reliable quantitation of major lipid components in a given lipid extract. On the downside, this method has a limited capability for detection of unexpected lipid species and is particularly vulnerable for overlapping isobaric compounds. In contrast to low resolution instruments, high resolution mass spectrometers deliver accurate mass and elemental composition of ions with very high confidence. Most lipid classes have an unambiguous fingerprint due to a certain and invariable number of the heteroatoms N, O, P and S. Due to this fact, the elemental composition of precursor and often also product ions contains highly valuable information about the lipid class. The Multiple Precursor Ion Scans (MPIS) method developed on a quadrupole-TOF instrument by the group of Shevchenko BIB012 BIB013 combines high resolution precursor ion scans on glycerophospholipid headgroups and fatty acyl moieties, resulting in the individual fatty acyl composition of glycerophospholipid species. The high resolving power is particularly helpful in the case of ambiguous product ions with mass differences in the first or second digit. The MPIS concept was successfully applied for quantitative global lipidome analysis in various cell systems including glycerophospholipids, glycerolipids, sphingolipids, sterols and various glycolipids BIB018 BIB019 BIB022 . The method has its limitations when one lipid class like triacylglycerol (TG) is present in bulk amounts and possibly suppresses ionization of other minor lipid classes BIB020 . A further development of the MPIS concept is shotgun lipidomics with a hybrid LTQ-Orbitrap instrument coupled to the Nanomate® ion source BIB014 . A schematic workflow of this platform is shown in Figure 1 . This system relies on accurate mass down to sub ppm range for both, precursor and product ions. With the recent development of higher energy collision-induced dissociation (HCD) even the low mass cut off problem of product ion spectra acquired in the LTQ could be overcome BIB023 . The Nanomate® provides plenty of time to be spent on each sample with only a few microliters of it consumed. This opens up the avenue for data-dependent acquisition of product ion spectra on all possible precursor ions, resulting in full scan precursor spectra and product ion spectra of literally every detectable lipid species at a resolution of 100,000 or more. Additionally, exact assignment of fatty acyl side chains can be achieved on a regular basis with this system. Quantitation is done by one internal standard per lipid class BIB021 , which is sufficient to compensate for varying ionization efficiencies. An interesting alternative to gas chromatography-mass spectrometry (GC-MS) analysis of fatty acids is published by the Welti group BIB015 . The CID-TOF system uses a quadrupole-TOF analyzer coupled to negative ESI direct infusion. Thereby mass selection in Q1 is turned off, Q2 fragments all ions and the TOF analyzer records intact fatty acid carboxylates with accurate mass. This provides the fingerprint of fatty acids including modified fatty acids without any prior derivatization step being necessary. Mentionable, this method only works for lipids which generate negative ions in ESI, but nevertheless comparison with GC-flame ionization detector (GC-FID) data shows good correlation BIB015 .