Shotgun lipidomics: Electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples

Abstract

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.

Alkaline methanolysis of lipid extracts extends shotgun lipidomics analyses to the low-abundance regime of cellular sphingolipids

“…For example, many lipid classes (e.g., sphingosine-1-phosphate, lysophosphatidic acid, acylcarnitine, etc.) can be efficiently extracted under acidic conditions [4]. Gangliosides and acylCoAs are highly soluble in polar solvents and are partitioned into the aqueous phase during chloroform extraction [17][18][19], which can be reverse-extracted by using butanol under acidic conditions.…”

“…Typically, a 1-min period of signal averaging in the profile mode was employed for each MS spectrum. For tandem mass spectrometry, the collision gas pressure was set at 1.0 mTorr but the collision energy varied with the classes of lipids as described previously [4,15]. Typically, a 2-to 5-min period of signal averaging in the profile mode was employed for each tandem MS spectrum.…”

“…We tested the reaction under different base conditions by using LiOH, NaOH, KOH, NaOMe, or LiOMe at various concentrations. We found that the free fatty acids generated from the treatment of LiOH, NaOH, or KOH could not be efficiently removed by washing with hexane due to a limited solubility of free fatty acids in hexane and that the quality of lipid analysis achieved using shotgun lipidomics might be affected when bases other than those containing lithium were used to treat lipid extracts [4]. The recovery of sphingosine was affected if ether was used to remove the free fatty acids generated.…”

“…In the future, research in lipidomics will expand to include the dynamics of lipidomes, subcellular organizations of lipidomes, and interactions of lipids with lipids, proteins, and other cellular moieties. Although lipidomics has emerged as a distinct field only within the past few years [1][2][3], numerous new discoveries and/or advances have already been made [3][4][5][6][7][8][9][10][11][12]. Its essential roles in identifying the biochemical mechanisms of lipid metabolism, investigating the functions of an individual gene of interest, identifying novel biomarkers, and evaluating drug efficacy, among others, are becoming increasingly recognized.…”

“…Second, some or all of these determined molecular species of the class are used as standards to determine the contents of other low-abundance or overlapping molecular species using one or multiple NL and/or PI scans as described above. Through this second step in the quantitation process, the linear dynamic range of quantitation can be dramatically extended by eliminating background noise and by filtering the overlapping molecular species through a multidimensional MS approach [4]. However, due to signal overlaps of lowabundance molecular species of a class of interest (e.g., sphingomyelin (SM)) with potential isomeric lipid molecular species in other lipid class(es) (e.g., choline glycerophospholipid (GPCho)) in both first and second steps of quantitation in shotgun lipidomics, identification and quantitation of these molecular species was previously inaccessible.…”