Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry in Lipidomics - - Chromatography Online
Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry in Lipidomics


LCGC North America
Volume 32, Issue 4, pp. 286-293

An analytical method for the global profiling of molecular lipids in biological samples, with particular emphasis on the plasmalogen lipids, is described. The global profiling method is based on ultrahigh-performance liquid chromatography combined with quadrupole time-of-flight-mass spectrometry (UHPLC–QTOF-MS). The profiling approach is complemented by UHPLC–LTQ-Orbitrap mass spectrometry in MS n mode for de novo lipid identification.



Lipids are an important class of essential metabolites and have many key biological functions. They are structural components of cell membranes, energy storage sources, and intermediates in signalling pathways (1,2). For example, tight control of membrane lipid composition is of central importance to maintain normal cellular physiology, and its dysregulation may affect membrane fluidity as well as topology, mobility, or activity of membrane-bound proteins. Lipids originate entirely or in part from two distinct types of building blocks: ketoacyl and isoprene groups. They are both functionally and structurally a very diverse group of compounds, partly because of the many possible variations of the lipid building blocks and the different ways of noncovalent linkage. The structural diversity of lipids is demonstrated by the huge number of molecular lipid species found in biological systems, which is estimated to be in the order of hundreds of thousands (3).


Figure 1: Structures of PC plasmalogen PC(p18:0/20:4) and corresponding PC(18:1/20:4).
Plasmalogens are a specific group of ether phospholipids. Structurally, they are glycerophospholipids which mainly consist of ethanolamine and choline, that is, phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) characterized by an alk-1'-enylether bond in position sn-1 where aliphatic moieties C16:0, C18:0, or C18:1 carbon chains are incorporated preferentially; the sn-2 position is specifically occupied by polyunsaturated fatty acids (Figure 1) (1–3). Plasmalogens are abundant lipid species representing 20% of the total pool of phospholipids in cells. They contribute to membrane structural integrity, and are also involved in multiple cellular functions such as vesicle formation and membrane fusion (4–6), ion transport (7,8) and generation of secondary signal mediators such as platelet activating factor (PAF) (9). The presence of the vinyl ether bond gives antioxidant properties to these molecules, which diminishes free radical–based cellular damage (10,11) and thus protects cells against oxidative stress. Indeed, plasmalogens have been shown to play an important role in human health as a factor involved in aging, obesity, diabetes, and diseases of the central nervous system (12–14).

Global characterization of lipids in biological samples — lipidomics — is a challenging task because of the high diversity of lipids. These approaches include shotgun lipidomics, which uses direct infusion of lipid extracts into the mass spectrometer (15,16), as well as liquid chromatography coupled to mass spectrometry (LC–MS) (17). While the shotgun approach is relatively simple and rapid, it suffers from matrix effects and ion suppression, and it is not very well suited for global profiling of previously unknown lipids. The advantage of LC–MS-based methods over the shotgun approach is the higher sensitivity as well as the ability, by using a nontargeted strategy, to detect and identify novel lipids. However, matrix effects cannot be fully avoided in LC–MS either, and careful optimization of eluent composition as well as clean-up steps is required to avoid carry-over and contamination.

Here, we describe an ultrahigh performance liquid chromatography–mass spectrometry (UHPLC–MS)-based global lipidomics platform and identification workflow for the characterization of plasmalogens from biological samples. In the identification stage, an LTQ-Orbitrap system (Thermo Fisher Scientific) is used as the detector for its outstanding mass accuracy and mass resolution. The ability to detect accurate masses allows the unequivocal compositional and structural elucidation of the compounds.


ADVERTISEMENT

blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:




 

LCGC COLUMNISTS 2014

Sample Prep Perspectives | Ronald E. Majors: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments.
LATEST: UV Detector Problems


Perspectives in Modern HPLC | Michael W. Dong: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Superficially Porous Particles: Perspectives, Practices, and Trends


MS — The Practical Art | Kate Yu: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis


LC Troubleshooting | John Dolan: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: Problems with Large-Molecule Separations


More LCGC Chromatography-Related Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>


Source: LCGC North America,
Click here