Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples. <s> BIB001 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> Neutral lipids are an important class of hydrophobic compounds found in all cells that play critical roles from energy storage to signal transduction. Several distinct structural families make up this class, and within each family there are numbers of individual molecular species. A solvent extraction protocol has been developed to efficiently isolate neutral lipids without complete extraction of more polar phospholipids. Normal-phase HPLC was used for the separation of cholesteryl esters (CEs), monoalkylether diacylglycerols, triacylglycerols, and diacylglycerols in a single HPLC run from this extract. Furthermore, minor lipids such as ubiquinone-9 could be detected in RAW 264.7 cells. Molecular species that make up each neutral lipid class can be analyzed both qualitatively and quantitatively by on-line LC-MS and LC-MS/MS strategies. The quantitation of >20 CE molecular species revealed that challenging RAW 264.7 cells with a Toll-like receptor 4 agonist caused a >20-fold increase in the content of CEs within cells, particularly those CE molecular species that contained saturated (14:0, 16:0, and 18:1) fatty acyl groups. Longer chain CE molecular species did not change in response to the activation of these cells. <s> BIB002 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> The sphingolipidome is the portion of the lipidome that encompasses all sphingoid bases and their derivatives. Whereas the most studied sphingoid base is sphingosine [(2S,3R,4E)-2-aminooctadecene-1,3-diol], mammals have dozens of structural variants, and hundreds of additional types have been found in other eukaryotic organisms and some bacteria and viruses. Multiplying these figures by the N-acyl-derivatives (“ceramides”) and the more than 500 phospho- and glyco- headgroups places the number of discrete molecular species in the tens of thousands or higher. Structure-specific, quantitative information about a growing fraction of the sphingolipidome can now be obtained using various types of chromatography coupled with tandem mass spectrometry, and application of these methods is producing many surprises regarding sphingolipid structure, metabolism, and function. Such large data sets can be difficult to interpret, but the development of tools that display results from genomic and lipidomic studies in a pathway relational, nodal, context can make it easier for investigators to deal with this complexity. <s> BIB003 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> Recently, it was reported that oxidized phosphatidylcholine shows biological activities via scavenger receptor CD36 or Toll-like receptor 4 (TLR4)-TRIF. Thus, the analysis of oxidized phospholipids is essential in understanding these biological roles. Here, we report an analytical method for oxidized phosphatidylcholines using multiple reaction monitoring (MRM) with theoretically expanded data sets. This analytical method was performed by a quadrupole linear ion trap mass spectrometer with ultra performance LC (UPLC). To investigate whether this established analytical method was applicable to biological samples, we performed variation analysis of oxidized PCs using a myocardial ischemia-reperfusion model. Most oxidized PCs were detected in higher amounts in the ischemic myocardium than in the non-ischemic myocardium. From these application results, this established method is a valuable tool for the global analysis of oxidized PCs. In the future, our study can provide further understanding of how oxidized phospholipids are produced and are correlated to various diseases. <s> BIB004 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules. <s> BIB005 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo2-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation. <s> BIB006 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> Recent rapid growth of lipidomics is mainly attributed to technological advances in mass spectrometry. Development of soft ionization techniques, in combination with computational tools, has spurred subsequent development of various methods for lipid analysis. However, none of these existing approaches can cover major cellular lipids in a single run. Here we demonstrate that a single method of liquid chromatography coupled with mass spectrometry (LCMS) can be used for simultaneous profiling of major cellular lipids including glycerophospholipids (PLs), sphingolipids (SPLs), waxes, sterols (ST) and mono-, di- as well as triacylglycerides (MAG, DAG, TAG). We applied this approach to analyze these lipids in various organisms including Saccharomyces cerevisiae and Schizosaccharomyces pombe. While phospholipids and triacylglycerides of S. pombe mainly contain 18 : 1 fatty acyls, those of S. cerevisiae contain 16 : 1, 16 : 0 and 18 : 1 fatty acyls. S. cerevisiae and S. pombe contain distinct sphingolipid profiles. S. cerevisiae has abundant inositol phytoceramides (IPC), while S. pombe contains high levels of free phytoceramides as well as short chain phytoceramides (t18:1/20 : 0-B) and IPC (t18:1/20 : 0-B). In S. cerevisiae, our results demonstrated accumulation of ergosterol esters in tgl1Delta cells and accumulation of various TAG species in tgl3Delta cells, which are consistent with the function of the respective enzymes. Furthermore, we, for the first time, systematically characterized lipids in S. pombe and measured their dynamic changes in Deltaplh1Deltadga1 cells at different growth phases. We further discussed dynamic changes of phospholipids, sphingolipids and neutral lipids in the progress of programmed cell death in Deltaplh1Deltadga1 cells of S. pombe. <s> BIB007 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> In the present work we describe the development of an analytical technique for simultaneous profiling of over 100 biochemically related lipid mediators in biological samples. A multistep procedure was implemented to extract eicosanoids and other bioactive lipids from the biological matrix, chromatographically separate them using fast reversed-phase liquid chromatography, tentatively identify new candidate eicosanoids through a matching process of retention times, isotope distribution patterns, and high-resolution orbitrap MS/MS fragmentation patterns, and subsequently quantify tentative candidates by means of analytical reference standards. Key new aspects of this profiling technique included the classification of bioactive lipids into 12 groups according to their calculated exact masses and the development of optimized liquid chromatographic conditions for these groups to achieve sufficient separation of the numerous isobaric and isomeric species, many of which exhibited virtually identical collision-ind... <s> BIB008 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> Sphingolipids comprise bioactive molecules which are known to play important roles both as intracellular and extracellular signalling molecules. Here we used a previously developed hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS) method to profile plasma sphingolipids. Method validation showed sufficient precision and sensitivity for application in large clinical studies. Sample stability testing demonstrated that immediate plasma separation is important to achieve reliable results. Analysis of plasma from 25 healthy blood donors revealed a comprehensive overview of free sphingoid base, sphingosylphosphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer), and ceramide-1-phosphate (Cer1P) species level. Besides the major sphingoid base sphingosine (d18:1), we found d16:1 and d18:2 species in most of these lipid classes. Interestingly, pronounced differences were detected in the species profiles of HexCer and LacCer. Additionally, sphingolipids were quantified in lipoprotein fractions prepared by fast performance liquid chromatography (FPLC). HexCer and LacCer showed similar distributions with about 50% in LDL, 40% in HDL and less than 10% in the VLDL fraction. More than 90% of sphingoid base phosphates were found in HDL and albumin containing fractions. In summary, HILIC-MS/MS provides a valuable tool to profile minor sphingolipid species in plasma and in lipoprotein fractions. Comparing profiles from tissues or blood cells, these species profiles may help to address the origin of plasma sphingolipids. <s> BIB009 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> By methylating the phosphate groups of PtdIns(3,4,5)P3 researchers can load this lipid more efficiently into a mass spectrometer and thus this lipid can be quantified in the presence of an internal synthetic standard. <s> BIB010 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> Oxysterols are present in mammalian brain at ng/g-μg/g levels while cholesterol is present at the mg/g level. This makes oxysterol analysis of brain challenging. In an effort to meet this challenge we have developed, and validated, an isolation method based on solid phase extraction and an analytical protocol involving oxidation/derivatisation (i.e., charge-tagging) followed by nano-flow liquid chromatography (nano-LC) combined with tandem mass spectrometry utilising multi-stage fragmentation (MS(n)). The oxidation/derivatisation method employed improves detection limits by two orders of magnitude, while nano-LC-MS(n) provides separation of isomers and allows oxysterol quantification. Using this method 13 different oxysterols have been identified in rat brain including 24S-hydroxycholesterol, 24S,25-epoxycholesterol and 7α,26-dihydroxycholest-4-en-3-one. The level of 24S-hydroxycholesterol in rat brain was determined to be 20.3±3.4 μg/g and quantitative estimates were made for the other oxysterols identified. The presence of a large excess of cholesterol over oxysterol in brain raises the problem of autoxidation during sterol isolation and sample preparation. Thus, in parallel to identification studies, the degree of cholesterol autoxidation occurring during sterol isolation and analysis has been evaluated with the aid of [(2)H(7)]-labelled cholesterol and cholesterol autoxidation products identified. <s> BIB011 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> Low Resolution Mass Spectrometry <s> Neutral lipids are a diverse family of hydrophobic biomolecules that have important roles in cellular biochemistry of all living species but have in common the property of charge neutrality. A large component of neutral lipids is the glycerolipids composed of triacylglycerols, diacylglycerols, and monoacylglycerols that can serve as cellular energy stores as well as signaling molecules. Another abundant lipid class in many cells is the cholesterol esters that are on one hand sterols and the other fatty acyl lipids, but in either case are neutral lipids involved in cholesterol homeostasis and transport in the blood. The analysis of these molecules in the context of lipidomics remains challenging because of their charge neutrality and the complex mixtures of molecular species present in cells. Various techniques have been used to ionize these neutral lipids prior to mass spectrometric analysis including electron ionization, atmospheric chemical ionization, electrospray ionization and matrix assisted laser desorption/ionization. Various approaches to deal with the complex mixture of molecular species have been developed including shotgun lipidomics and chromatographic-based separations such as gas chromatography, reversed phase liquid chromatography, and normal phase liquid chromatography. Several applications of these approaches are discussed. . <s> BIB012
The invention of ESI enabled coupling of HPLC with mass spectrometry in a highly efficient manner for the first time  . This instrumental combination opened up completely new analytical perspectives in lipid research by combining the separation power of HPLC with the selectivity of mass spectrometry. Complex lipid classes like glycerolipids, glycerophospholipids or even glycolipids were analytically amenable on a regular basis by chromatography coupled to mass spectrometry, now termed LC-MS. Compared to direct infusion systems HPLC adds retention time as another layer of selectivity. On one hand this results in increased specificity for lipid identification, but on the other hand it complicates quantitation, because every spectrum in an LC-MS run has to be regarded as a single event with unique matrix effects and solvent composition ( Figure 2 ). Therefore quantitative aspects are generally more difficult to be standardized than for direct infusion methods. Excellent examples of lipidomic LC-MS analysis have recently been shown for human plasma BIB005 and subcellular organelle lipidomics of TLR-4 activated macrophages BIB006 by the LIPID MAPS consortium. These publications show very well that the whole lipidome of an organism or tissue cannot be determined by a single experimental setup but rather by a combination of various methods, most of them LC-MS based. The fastest and simplest way to monitor changes in a lipid profile by LC-MS is LC/single ion monitoring (SIM)/MS BIB007 . This method is based on ESI full scan quadrupole MS of intact molecular adduct ions. Plotting of retention time versus m/z and intensity provides a 3D lipidomic fingerprint of a sample, which can be used to monitor changes between statistical groups by differential profiles in a fast and comprehensive manner. A very efficient way to maximize sensitivity is targeted lipidomics with HPLC-triple quadrupole instrumentation in MRM mode. Due to the instrument's high dynamic range and the selectivity of retention time in conjunction with known precursor / product ion pairs, it is the method of choice for lipid quantitation. Recent developments in scan speed of triple quadrupole mass spectrometers result in a duty cycle of up to 100 Hz, which provides the basis for fast and reliable quantitation of whole lipid classes within one chromatographic run BIB008 BIB009 BIB003 . In some rare cases, molecular species of lipids are composed of exactly the same building blocks, resulting in the same elemental composition and the same fragments, which renders MRM analysis basically useless. In this case, good chromatographic separation is mandatory as shown for bis(monoacylglycero)phosphate (BMP) and cardiolipin (CL) by the group of Liebisch BIB009 . A good compromise between targeted and non-targeted analysis on a triple quadrupole instrument are precursor ion-and constant neutral loss scans. Although sensitivity for such scans might not be as high as in MRM mode, it opens the possibility to find unexpected species within the lipid classes surveyed by the respective precursor or constant neutral loss scans. Such systems are quite frequently used with ESI and reversed phase HPLC coupling. An excellent example of this technique is shown by Retra et al.  . The applied very shallow 60 min gradient results in baseline separation of glycerophospholipid species. The use of constant neutral loss scans for phosphatidylinositol phosphate (PIP) identification and quantitation is shown by Clark et al. BIB010 . Identification of lipid molecular species with low resolution instrumentation is best achieved by MS n analysis, because this kind of analysis provides a high degree of structural information. The preferred instrumentation is either (linear) ion trap or triple quadrupole technology. Usually acquisition time for a certain compound is limited in LC-MS approaches and, as acquisition of product ion spectra takes much longer than for MRM spectra, the number of lipids to be covered is a potential bottleneck. Nevertheless, in cases of a defined number of lipids, this can be a highly specific identification strategy as shown successfully for oxysterols BIB011 , positional isomer analysis of phospholipids BIB004 and in depth analysis of PIP species BIB001 . While chromatographic separation of lipids is often performed on reversed phase HPLC according to fatty acyl chains, this strategy runs into its limits when cholesterol esters (CE) are to be analyzed in the presence of bulk amounts of TG. Due to their very similar hydrophobicity CE and TG are hardly separated on reversed phase and hydrophilic interaction liquid chromatography (HILIC) columns, resulting in suppression of low abundant CE by TG (Figure 2 ). In contrast, silica-based normal phase HPLC provides separation of these lipid classes by their polar functional groups, but usually highly non polar solvents with low ionization capacity have to be used. Hutchins et al. BIB002 use APCI and post-column addition of a polar solvent to increase ionization properties of the non polar solvent eluting from normal phase HPLC. This results in a practicable online bridging between normal phase HPLC and triple quadrupole mass spectrometry, which can either be used in precursor ion, MRM or single quadrupole mode for determination of neutral lipids BIB005 BIB012 .