Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics has undergone dramatic developments over the past decade. This review focuses on state of the art in LC-MS-based lipidomics, covering all the steps of global lipidomic profiling. By reviewing 185 original papers and application notes, we can conclude that current advanced LC-MS-based lipidomics methods involve: (1) lipid extraction schemes using chloroform/MeOH or methyl tert -butyl ether (MTBE)/MeOH, both with addition of internal standards covering each lipid class; (2) LC separation of lipids using short microbore C18 or C8 columns with sub-2-µm or 2.6–2.8-µm (fused-core) particle size with analysis time (3) electrospray ionization in positive- and negative-ion modes with full spectra acquisition using high-resolution MS with capability to MS/MS. Phospholipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, phosphatidylglycerols) followed by sphingomyelins, di- and tri-acylglycerols, and ceramides were the most frequently targeted lipid species.

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Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry

Understanding how biological molecules are generated, metabolized and eliminated in living systems is important for interpreting processes such as immune response and disease pathology. While genomic and proteomic studies have provided vast amounts of information over the last several decades, interest in lipidomics has also grown due to improved analytical technologies revealing altered lipid metabolism in type 2 diabetes, cancer, and lipid storage disease. Mass spectrometry (MS) measurements are currently the dominant approach for characterizing the lipidome by providing detailed information on the spatial and temporal composition of lipids. However, interpreting lipids' biological roles is challenging due to the existence of numerous structural and stereoisomers (i.e. distinct acyl chain and double-bond positions), which are often unresolvable using present approaches. Here we show that combining liquid chromatography (LC) and structurally-based ion mobility spectrometry (IMS) measurement with MS analyses distinguishes lipid isomers and allows insight into biological and disease processes.

/pdf/uncovering-biologically-significant-lipid-isomers-with-2duxeq3enr.pdf

Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry

■ CONTENTS Analytical Methods of Lipidomics 162 Extraction Methods of Lipids 163 Direct-Infusion MS-Based Lipidomics Analysis 163 Electrospray Ionization (ESI) MS 163 Matrix-Assisted Laser Desorption Ionization (MALDI) MS 164 Other Ionization Techniques Used in Lipidomics Analysis 164 Mass Spectrometry Imaging (MSI) in Lipidomics 165 Chromatography-Based Lipidomics Analysis 165 TLC 166 GC 166 HPLC 166 SFC 167 CE 167 Spectroscopy 167 Nuclear Magnetic Resonance (NMR) 167 Raman Spectroscopy 168 Applications of Lipidomics Analysis in Life Science 168 Lipidomics of Cancers 168 Lipidomics of Alzheimer’s Disease (AD) 168 Cellular Lipidomics 168 Plants 168 Conclusions 170 Author Information 170 Corresponding Author 170 Notes 170 Biographies 170 Acknowledgments 171 References 171

Analytical methods in lipidomics and their applications.

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor onto bacterial cell membranes. These proteins play important roles in a wide variety of bacterial physiological processes, including virulence, and induce innate immune reactions by functioning as ligands of the mammalian Toll-like receptor 2. We review recent advances in our understanding of bacterial lipoprotein structure, biosynthesis and structure–function relationships between bacterial lipoproteins and Toll-like receptor 2. Notably, 40 years after the first report of the triacyl structure of Braun's lipoprotein in Escherichia coli, recent intensive MS-based analyses have led to the discovery of three new lipidated structures of lipoproteins in monoderm bacteria: the lyso, N-acetyl and peptidyl forms. Moreover, the bacterial lipoprotein structure is considered to be constant in each bacterium; however, lipoprotein structures in Staphylococcus aureus vary between the diacyl and triacyl forms depending on the environmental conditions. Thus, the lipidation state of bacterial lipoproteins, particularly in monoderm bacteria, is more complex than previously assumed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/febs.12041

Lipoproteins in bacteria: structures and biosynthetic pathways.

High throughput quantitative molecular lipidomics

Ozone-induced dissociation (OzID) exploits the gas-phase reaction between mass-selected lipid ions and ozone vapor to determine the position(s) of unsaturation In this contribution, we describe the modification of a tandem linear ion-trap mass spectrometer specifically for OzID analyses wherein ozone vapor is supplied to the collision cell This instrumental configuration provides spatial separation between mass-selection, the ozonolysis reaction, and mass-analysis steps in the OzID process and thus delivers significant enhancements in speed and sensitivity (ca 30-fold) These improvements allow spectra revealing the double-bond position(s) within unsaturated lipids to be acquired within 1 s significantly enhancing the utility of OzID in high-throughput lipidomic protocols The stable ozone concentration afforded by this modified instrument also allows direct comparison of relative reactivity of isomeric lipids and reveals reactivity trends related to (1) double-bond position, (2) substitution position on the glycerol backbone, and (3) stereochemistry For cis- and trans-isomers, differences were also observed in the branching ratio of product ions arising from the gas-phase ozonolysis reaction, suggesting that relative ion abundances could be exploited as markers for double-bond geometry Additional activation energy applied to mass-selected lipid ions during injection into the collision cell (with ozone present) was found to yield spectra containing both OzID and classical-CID fragment ions This combination CID-OzID acquisition on an ostensibly simple monounsaturated phosphatidylcholine within a cow brain lipid extract provided evidence for up to four structurally distinct phospholipids differing in both double-bond position and sn-substitution U Am Soc Mass Spectrom 2010, 21, 1989-1999) (C) 2010 American Society for Mass Spectrometry

Ozone-Induced Dissociation on a Modified Tandem Linear Ion-Trap : Observations of Different Reactivity for Isomeric Lipids

Ozone-induced dissociation (OzID) is an alternative ion activation method that relies on the gas phase ion-molecule reaction between a mass-selected target ion and ozone in an ion trap mass spectrometer. Herein, we evaluated the performance of OzID for both the structural elucidation and selective detection of conjugated carbon-carbon double bond motifs within lipids. The relative reactivity trends for \[M + X](+) ions (where X = Li, Na, K) formed via electrospray ionization (ESI) of conjugated versus nonconjugated fatty acid methyl esters (FAMEs) were examined using two different OzID-enabled linear ion-trap mass spectrometers. Compared with nonconjugated analogues, FAMEs derived from conjugated linoleic acids were found to react up to 200 times faster and to yield characteristic radical cations. The significantly enhanced reactivity of conjugated isomers means that OzID product ions can be observed without invoking a reaction delay in the experimental sequence (i.e., trapping of ions in the presence of ozone is not required). This possibility has been exploited to undertake neutral-loss scans on a triple quadrupole mass spectrometer targeting characteristic OzID transitions. Such analyses reveal the presence of conjugated double bonds in lipids extracted from selected foodstuffs. Finally, by benchmarking of the absolute ozone concentration inside the ion trap, second order rate constants for the gas phase reactions between unsaturated organic ions and ozone were obtained. These results demonstrate a significant influence of the adducting metal on reaction rate constants in the fashion Li > Na > K.

Ozone-induced dissociation of conjugated lipids reveals significant reaction rate enhancements and characteristic odd-electron product ions

Methods and systems for performing mass spectrometry are provided herein. The method includes ionizing one or more compounds to generate ions, passing the ions into an ion mobility spectrometer, providing ozone, reacting the ions with ozone to produce ozone-induced fragment ions, and performing mass analysis and detection of the ozone-induced fragment ions. The system can comprise an ion source for ionizing one or more compounds to generate ions, an ion mobility spectrometer for receiving and separating the ions, an ozone supply for introducing ozone into the system; a reaction region for reacting the ions with the ozone to produce ozone- induced fragment ions, and a mass spectrometer for performing mass analysis and detection of the ozone-induced fragment ions.

Method and apparatus for ion mobility spectrometry

Methods and apparatus for processing fluids on a macro- or micro-scale are described. In various aspects, a fluid may have a plurality of elongated (i.e., substantially rod-shaped) magnetic elements disposed therein within a fluid container. An illustrative fluid container is an actuator electrode or a processing vial of a microfluidic device, such as a digital microfluidic device. A magnet component may be configured to generate a magnetic force sufficient to influence the movement of the plurality of elongated magnetic elements within the fluid to be processed. For example, the magnetic force (or magnetic force gradient) may influence the plurality of elongated magnetic elements to rotate, spin, and/or move laterally side-to-side. The shape and movements of the plurality of elongated magnetic elements facilitate the rapid and efficient processing of the fluid, such as fluid mixing and/or fluid separation.

Magnetic Elements for Processing Fluids

An electrospray ion source method and system is provided for detecting emitter failure comprising a liquid chromatography column suitable for chromatographic separation of a sample. The column can have an inlet for receiving the sample; and an outlet for ejecting the sample. A make-up flow channel is provided for introducing make-up flow of liquid to the sample post-column, wherein the make-up flow normalizes the spray current. An electrospray ionization source is provided having one or more electrospray ionization emitter nozzles for receiving the make-up flow containing sample. A power supply can provide a voltage to the one or more emitter nozzles, and a measurement device can measure and monitor the spray current.

John Lawrence Campbell

Papers

Ozone-Induced Dissociation on a Modified Tandem Linear Ion-Trap : Observations of Different Reactivity for Isomeric Lipids

Ozone-induced dissociation of conjugated lipids reveals significant reaction rate enhancements and characteristic odd-electron product ions

Method and apparatus for ion mobility spectrometry

Magnetic Elements for Processing Fluids

Method and system for introducing make-up flow in an electrospray ion source system