Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes. Recent developments in mass spectrometric technology enabled the comprehensive characterization of eukaryotic lipidomes, fostering the molecular biology of lipids and metabolism-related disorders (reviewed in Refs. 1-4). Typically, lipidome profiling by mass spectrometry proceeds along LC-MS or shotgun approaches. The former identifies and quantifies lipid species preseparated by normal or reversed-phase chromatography coupled online to a mass spectrometer, which is capable of fast acquisition of MS or MS/MS spectra (5-8). In contrast, in shotgun lipidomics, total lipid extracts are infused directly into a mass spectrometer, and the molecular characterization of lipid species relies either on the accurately determined m/z of precursor ions (9) or on the detection of specific fragment ions or neutral losses in tandem mass spectrometric experiments (1, 9-12).Regardless of the analytical approach used, its success depends on the completeness of the extraction of lipids from corresponding cells, fluids, or tissues. Lipids of all major classes could be recovered via chloroform/methanol extraction, typically according to the Folch, Lees, and Sloane Stanley (13) or Bligh and Dyer (14) recipes (15), in which they are mostly enriched in the chloroform phase.Electrospray mass spectrometry, a major tool for analyzing complex lipidomes, is particularly sensitive towards the quality of lipid extracts. Coextracted components of biological matrix and salts (often, without further definition, termed background) affect both the sensitivity and specificity of lipid analysis. Often, abundant background ions obscure lipid precursors, and their MS/MS spectra are densely populated with "ghost" peaks and abundant chemical noise. Adducts with common background cations (sodium, potassium) and anions (chloride) increase the ambiguity of molecular species assignment and affect the accuracy of quantitative determination.Because of the higher density of chloroform compared with a water/methanol mixture, it forms the lower phase of the two-phase partitioning system. While collecting the chloroform fraction, a glass pipette or a needle of the pipetting robot reaches it through a voluminous layer...

Nomenclature Committee (ILCNC) in 2005 ( 1 ) and updated in 2009 ( 2 ). This system places lipids into eight categories and is available online on the LIPID MAPS website (http://www.lipidmaps.org). The LIPID MAPS nomenclature precisely describes lipid structures.The key technology for lipid species analysis is mass spectrometry (MS) ( 3, 4 ). Typically, MS analysis without intermediate chemical steps does not provide the structural details covered by the LIPID MAPS nomenclature, which led mass spectrometrists to use a variety of different notations for lipid species. Moreover, lipid species are frequently annotated based on assumptions. For example, a precursor ion scan of m/z 184, a standard approach to detect phosphatidylcholine (PC) and sphingomyelin (SM) species, is neither able to differentiate PC species containing an ether bond from diacyl species ( Fig. 1A ), nor is it able to differentiate the sphingoid base in SM ( 5 ). Similarly, annotation of phosphatidylethanolamine (PE) species, particularly plasmalogens, should not be based on head group-specifi c positive neutral loss (NL 141 ) or negative precursor ion (PIS m/z 196) scans ( 6 ), which identify only the lipid class but do not provide specifi c mass spectrometric analysis ( 7 ) ( Fig. 1B ).Although lipidologists possess a "biological intelligence," which allows them to interpret MS data in a manner that recognizes what is likely or not likely to be the correct structure of a particular lipid species, we think there is need for a standardized, practical shorthand notation of lipid structures derived from MS approaches that enables correct and concise reporting of data and their deposition in databases. Our proposal is based on common, offi cially accepted terms and on the LIPID MAPS terminology. In addition, it takes Abstract There is a need for a standardized, practical annotation for structures of lipid species derived from mass spectrometric approaches; i.e., for high-throughput data obtained from instruments operating in either high-or lowresolution modes. This proposal is based on common, offi cially accepted terms and builds upon the LIPID MAPS terminology. It aims to add defi ned levels of information below the LIPID MAPS nomenclature, as detailed chemical structures, including stereochemistry, are usually not automatically provided by mass spectrometric analysis. To this end, rules for lipid species annotation were developed that refl ect the structural information derived from the analysis. For example, commonly used head group-specifi c analysis of glycerophospholipids (GP) by low-resolution instruments is neither capable of differentiating the fatty acids linked to the glycerol backbone nor able to defi ne their bond type (ester, alkyl-, or alk-1-enyl-ether). This and other missing structural information is covered by the proposed shorthand notation presented here. Beyond GPs, we provide shorthand notation for fatty acids/acyls (FA), glycerolipids (GL), sphingolipids (SP), and sterols (ST). In summary, this defi ned shorthand nomenclatur...

Excessive uptake of atherogenic lipoproteins such as modified lowdensity lipoprotein complexes by vascular macrophages leads to foam cell formation, a critical step in atherogenesis. Cholesterol efflux mediated by high-density lipoproteins (HDL) constitutes a protective mechanism against macrophage lipid overloading. The molecular mechanisms underlying this reverse cholesterol transport process are currently not fully understood. To identify effector proteins that are involved in macrophage lipid uptake and release, we searched for genes that are regulated during lipid influx and efflux in human macrophages using a differential display approach. We report here that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is induced in monocyte-derived macrophages during cholesterol influx mediated by acetylated low-density lipoprotein. Conversely, lipid efflux in cholesterol-laden macrophages, mediated by the cholesterol acceptor HDL 3, suppresses the expression of ABCG1. Immunocytochemical and flow cytometric analyses revealed that ABCG1 is expressed on the cell surface and in intracellular compartments of cholesterol-laden macrophages. Inhibition of ABCG1 protein expression using an antisense strategy resulted in reduced HDL 3-dependent efflux of cholesterol and choline-phospholipids. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified an additional set of ABC genes whose expression is regulated by cholesterol uptake or HDL 3-mediated lipid release, suggesting a potential function for these transporters in macrophage lipid homeostasis. Our results demonstrating a regulator function for ABCG1 in cholesterol and phospholipid transport define a biologic activity for ABC transporters in macrophages.