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Shotgun Lipidomics is a robust methodology for the characterization of the lipidome of complex biological samples. This assay is among the most quantitative lipidomics methods and is capable of surveying a wide breadth of lipid subclasses, both neutral and polar. The shortfalls of the technique include limitations in lipid species characterization and computationally demanding data analysis requiring isotopic and isobaric overlap correction. Differential Mobility Spectrometry (DMS) has demonstrated its utility in enabling acyl tail characterization within a Shotgun Lipidomics experiment. Here, we present a workflow for DMS Shotgun Lipidomics that measures 1400 possible lipid species. It utilizes the Shotgun Lipidomics Assistant (SLA) application, an open-source application that supervises the data analysis for an expansive Shotgun Lipidomics experiment., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lipids serve an essential role in multiple cellular functions including signaling, metabolism, energy storage, and membrane constitution. Lipidomics, the study of lipids using analytical chemistry, allows for the study of disease states and cellular metabolism. Shotgun lipidomics is a technique that involves direct-infusion electrospray ionization (ESI) and analysis with a triple quadrupole mass spectrometer. Triple quadrupole mass spectrometry is ideally suited for lipidomics analysis because it allows for class-specific identification of lipids. Individual lipid class can be identified by the adjustment of three parameters-collision energy, ion mode, and scan type. This chapter describes the use of a triple quadrupole mass spectrometer, the TSQ Quantum Access MAX, to perform lipidomics analysis with high sensitivity, accuracy, and precision., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The emerging field of lipidomics presents the systems biology approach to identify and quantify the full lipid repertoire of cells, tissues, and organisms. The importance of the lipidome is demonstrated by a number of biological studies on dysregulation of lipid metabolism in human diseases such as cancer, diabetes, and neurodegenerative diseases. Exploring changes and regulations in the huge networks of lipids and their metabolic pathways requires a lipidomics methodology: advanced mass spectrometry that resolves the complexity of the lipidome. Here, we report a comprehensive protocol of quantitative shotgun lipidomics that enables identification and quantification of hundreds of molecular lipid species, covering a wide range of lipid classes, extracted from cultured mammalian cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

The precorneal tear film keeps the eye surface moist and helps to maintain normal eye function. The outermost lipid layer of the tear film, which attenuates tear film evaporation, contains meibum secreted from the meibomian gland. Most meibum lipids are neutral, including wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs), along with some polar lipids including free fatty acids (FFAs), O-acyl-ω-hydroxy fatty acids (OAHFAs), and trace phospholipids. Detection of neutral lipids by mass spectrometry (MS) is challenging due to interference from impurities, particularly when working with minute-volume meibum samples. Here, we describe procedures for sample preparation and MS analysis of these elusive meibum lipids that can be used to examine dry eye disease mechanisms. Because the method described here minimizes impurity peaks for lipids generally, neutral and otherwise, it may be applied to high-sensitivity analysis of other biological samples.

Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts, lipid rafts isolated by methods that use detergents frequently yield different results. Artifacts can also be introduced through the use of detergents. These limitations could be overcome with a detergent-free method which eliminates possible artificial influences. Importantly, lipid rafts prepared with a detergent-free method is more compatible to mass spectrometric analysis since the ion suppression effect is largely reduced.This chapter describes a detergent-free two-step method for preparation of lipid rafts. Firstly, a purified plasma membrane fraction is prepared from cells by sedimentation of the postnuclear supernatant (PNS) in a Percoll gradient. Secondly, the as-prepared plasma membranes are sonicated to release lipid rafts which are further isolated by flotation in a continuous gradient of Optiprep solution. Then, we introduce a typical shotgun lipidomics workflow that can be used as a cost-effective and relatively high throughput method to determine the lipidomes of lipid rafts.The method also makes an easy start for lipidomics studies.

Lipidomics approaches provide quantitative characterization of hundreds of lipid species from biological samples. Recent studies highlight the value of these methods in studying circadian biology, and their potential goes far beyond studying lipid metabolism per se. For example, lipidomics analyses of subcellular compartments can be used to determine daily rhythmicity of different organelles and their intracellular dynamics. In this chapter we describe in detail the procedure for around the clock shotgun lipidomics, from sample preparation to bioinformatics analyses. Sample preparation includes biochemical fractionation of nuclei and mitochondria from mouse liver harvested throughout the day. Lipid content is determined and quantified, in unbiased manner and with wide coverage, using multidimensional mass spectrometry shotgun lipidomics (MDMS-SL). Circadian parameters are then determined with nonparametric statistical tests. Overall, the approach described herein is applicable for various animal models, tissues, and organelles, and is expected to yield new insight on various aspects of circadian biology and lipid metabolism.

Over the last few decades, MS-based lipidomics has emerged as a powerful tool to study lipids in biological systems. This success is driven by the constant demand for complete and reliable data. The improvement of MS-based lipidomics will continue to be dependent on the advances in the technology of mass spectrometry and related techniques including separation and bioinformatics, and more importantly, on gaining insight into the knowledge of lipid chemistry essential to develop methodology for lipid analysis. It is hoped that the protocols in this book, collected from experts in their fields, can offer the beginner and the advanced user alike, useful tips toward successful lipidomic analysis.

Lipidomics is a powerful approach that can provide quantitative characterization of hundreds of lipid species from biological samples. Recent studies have highlighted the value of lipidomics to study myelin biology. This chapter provides a detailed description for the application of multidimensional mass spectrometry shotgun lipidomics (MDMS-SL) to neuroscience research and particularly to the analysis of brain lipidomes with a particular emphasis on myelin lipids, from sample preparation to bioinformatics analyses. Sample preparation includes brain sample harvesting, homogenization, and lipid extraction. Lipid content is determined and quantified, in an unbiased manner and with wide coverage, using MDMS-SL. Overall, the approach described herein is applicable for whole brain tissue or specific brain regions (e.g., hippocampus, cerebellum), and is expected to yield new insights on various aspects of myelin biology and lipid metabolism.

This protocol provides a step-by-step guide to shotgun sphingolipid analysis of aqueous humor. We describe the Bligh and Dyer crude lipid extraction method and electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled with MZmine 2.21 data processing for identification and ratiometric quantitation of sphingosine, sphingosine-1-phosphate, sphingomyelin, and ceramide.

Shotgun lipidomics offers fast and reproducible identification and quantification of lipids in clinical samples. Lipid extraction procedures based on the methyl tert-butyl protocol are well established for performing shotgun lipidomics in biomedical research. Here, we describe a shotgun lipidomics workflow that is well suited for the analysis of clinical samples such as tissue samples, blood plasma, and peripheral blood mononuclear cells.

Recent studies have illuminated novel roles of lysosomes that go far beyond simple catabolism and function in the coordination of cellular metabolism and signaling. Promising therapeutic strategies emerge from knowledge in the molecular mechanisms and physiological roles of lipid metabolism in lysosomes. Global monitoring of the function and dysregulation of lysosomal lipid metabolism requires a methodology that resolves the complexity of lysosomal lipidome by quantitatively detecting hundreds of lipid species of diverse physicochemical properties. We describe here a detailed protocol that couples isolation of superparamagnetic iron dextran-loaded lysosomes from cultured mammalian cell lines with quantitative mass spectrometry-based shotgun lipidomics.

Clinical lipidomics is an emerging biomarker discovery approach that compares lipid profiles under pathologically and physiologically normal conditions. Here we describe a method for the absolute (molar) quantification of more than 200 molecules from 14 major lipid classes from 5 μL of human blood plasma using high-resolution top-down shotgun mass spectrometry. Because of its technical simplicity and robustness, the protocol lends itself for high-throughput clinical lipidomics screens.

The emerging field of lipidomics presents the systems biology approach to identify and quantify the full lipid repertoire of cells, tissues, and organisms. The importance of the lipidome is demonstrated by a number of biological studies on dysregulation of lipid metabolism in human diseases such as cancer, diabetes, and neurodegenerative diseases. Exploring changes and regulations in the huge networks of lipids and their metabolic pathways requires a lipidomics methodology: Advanced mass spectrometry that resolves the complexity of the lipidome. Here, we report a comprehensive protocol of quantitative shotgun lipidomics that enables identification and quantification of hundreds of molecular lipid species, covering a wide range of lipid classes, extracted from cultured mammalian cells.