Lipidomics, after genomics and proteomics, is a newly and rapidly expanding research field that studies cellular lipidomes and the organizational hierarchy of lipid and protein constituents mediating life processes. Lipidomics is greatly facilitated by recent advances in, and novel applications of, electrospray ionization mass spectrometry (ESI/MS). In this review, we will focus on the advances in ESI/MS, which have facilitated the development of shotgun lipidomics and the utility of intrasource separation as an enabling strategy for utilization of 2D mass spectrometry in shotgun lipidomics of biological samples. The principles and experimental details of the intrasource separation approach will be extensively discussed. Other ESI/MS approaches towards the quantitative analyses of global cellular lipidomes directly from crude lipid extracts of biological samples will also be reviewed and compared. Multiple examples of lipidomic analyses from crude lipid extracts employing these approaches will be given to show the power of ESI/MS techniques in lipidomics. Currently, modern society is plagued by the sequelae of lipid-related diseases. It is our hope that the integration of these advances in multiple disciplines will catalyze the development of lipidomics, and such development will lead to improvements in diagnostics and therapeutics, which will ultimately result in the extended longevity and an improved quality of life for humankind.

Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base searches for proteins containing calcium-independent phospholipase A 2 (iPLA 2 ) dual signature nucleotide ((G/A)XGXXG) and lipase (GX-SXG) consensus sequence motifs identified a novel subfamily of three putative iPLA 2 /lipase family members designated iPLA 2 ⑀, iPLA 2 , and iPLA 2 (previously named adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA 2 subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA 2 ⑀, iPLA 2 , and iPLA 2 also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, a mechanism-based suicide substrate inhibitor of all known iPLA 2 s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC 50 ‫؍‬ 0.1-0.5 M). Quantitative PCR revealed dramatically increased expression of iPLA 2 ⑀ and iPLA 2 transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA 2 isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.Obesity and its associated clinical sequelae (e.g. type 2 diabetes, atherosclerosis, and hypertension) represent the major and most rapidly expanding health epidemic in industrialized nations (1-4). Obesity results from an abnormal increase in white adipose tissue mass, primarily in the form of triglycerides, and in humans is thought to be caused by a complex array of genetic, environmental, and hormonal factors (1, 4). Under conditions of obesity, serum nonesterified fatty acids are elevated, contributing to the accumulation of triglycerides in nonadipose tissues (e.g. hepatic, myocardial, and pancreatic) and to the development of the type 2 metabolic syndrome (5). The combined effects of excess cellular triglycerides, fatty acylCoAs, and free fatty acids are believed to be primary mediators of the lipotoxic effects of obesity that include decreased insulin sensitivity, increased oxidative stress, reduced metabolic capacity, and increased rates of cellular apoptosis in multiple organ systems (5-8).Triacylglycerol/fatty acid recycling is an important mechanism by which adipocytes modulate fatty acyl flux in response to changing metab...

Lipidomics is a rapidly expanding research field in which multiple techniques are utilized to quantitate the hundreds of chemically distinct lipids in cells and determine the molecular mechanisms through which they facilitate cellular function. Recent developments in electrospray ionization mass spectrometry (ESI/MS) have made possible, for the first time, the precise identification and quantification of alterations in a cell's lipidome after cellular perturbations. This review provides an overview of the essential role of ESI/MS in lipidomics, presents a broad strategy applicable for the generation of lipidomes directly from cellular extracts of biological samples by ESI/MS, and summarizes salient examples of strategies utilized to conquer the lipidome in physiologic signaling as well as pathophysiologically relevant disease states. Because of its unparalleled sensitivity, specificity, and efficiency, ESI/MS has provided a critical bridge to generate highly accurate data that fingerprint cellular lipidomes to facilitate insight into the functional role of subcellular membrane compartments and microdomains in mammalian cells. We believe that ESI/MS-facilitated lipidomics has now opened a critical door that will greatly increase our understanding of human disease. -Han, X., and R. W. Gross. Lipidomics is a rapidly expanding research field fueled by recent advances in, and novel applications of, electrospray ionization mass spectrometry (ESI/MS). Lipidomics is focused on identifying alterations in lipid metabolism and lipid-mediated signaling processes that regulate cellular homeostasis during health and disease. Research in lipidomics incorporates multiple techniques to quantify the precise chemical constituents in a cell's lipidome, identify their cellular organization (subcellular membrane compartments and domains), delineate the biochemical mechanisms through which lipids interact with each other and with crucial membrane-associated proteins, determine lipid-lipid and lipid-protein conformational space and dynamics, and quantify alterations in lipid constituents after cellular perturbations. Through the detailed quantification of a cell's lipidome (e.g., lipid classes, subclasses, and individual molecular species), the kinetics of lipid metabolism, and the interactions of lipids with cellular proteins, lipidomics has already provided new insights into health and disease. The true power and promise of lipidomics, however, is only beginning to be realized.Decades of painstaking research in the 1970s and 1980s developed a straight and reversed-phase HPLC system that could "rapidly" (30 min) separate phospholipid classes, molecular species, and regioisomers into individual chemical constituents (1-6). These techniques, however, were labor intensive and required sensitive separation methods for which quantitation was difficult, because the → * transition during UV detection was not strictly proportional to mass content (7). Moreover, these procedures could not meet the needs for the application of lipidomi...

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.

This report presents the strategies underlying the automated identification and quantification of individual lipid molecular species through array analysis of multi-dimensional mass spectrometrybased shotgun lipidomics (MDMS-SL) data which are acquired directly from lipid extracts after direct infusion and intrasource separation. The automated analyses of individual lipid molecular species in the program employ a strategy where MDMS-SL data from building block analyses using precursor-ion and/or neutral loss scans are used to identify individual molecular species followed by quantitation. Through this strategy, the program screens and identifies species in a high throughput fashion from a built-in database of over 36,000 potential lipid molecular species constructed employing known building blocks. The program then uses a two-step procedure for quantitation of the identified species possessing a linear dynamic range over three orders of magnitude and re-verifies the results when necessary through redundant quantification of multi-dimensional mass spectra. This program is designed to be easily adaptable for other shotgun lipidomics approaches which are currently used for mass spectrometric analysis of lipids in the field. Accordingly, the development of this program should greatly accelerate high throughput analysis of lipids using MDMS-based shotgun lipidomics. KeywordsAutomation; high throughput; intrasource separation; lipidomics; multidimensional mass spectrometry; shotgun lipidomicsIn the current practice of lipidomics, multi-dimensional mass spectrometry (MDMS)-based shotgun lipidomics (MDMS-SL) 1-3 is a well-recognized platform to analyze individual lipid molecular species directly from lipid extracts of biological samples. Through exploiting the unique chemistries of different lipid classes in MDMS-SL the need for chromatography is largely replaced for even extremely low abundant molecular species. For example, differential hydrophobicity and differential sensitivity to base treatment are exploited during a multiplexed extraction approach 4 . Through exploiting the differential electrical properties of lipid classes to selectively ionize a certain category of lipid classes under multiplexed experimental The unique chemical structure of the majority of lipid molecular species is comprised of linear combinations of a small number of building blocks that include backbones, head groups, and aliphatic chains 1,6 . We have exploited these building blocks through the use of two powerful tandem MS techniques (i.e., neutral loss scan (NLS) and precursor-ion scan (PIS)) in a massramp fashion and developed MDMS to identify individual lipid molecular species 1,7 . MDMS is totally analogous to multi-dimensional nuclear magnetic resonance (NMR) spectroscopy except that axes of mass in MDMS instead of frequency in NMR are routinely used 1 . In MDMS, the use of crossed peaks of the scanned fragments (i.e., lipid building blocks) with any given molecular ion allows us to determine the molecular identities of ions ...