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 MSn 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.
Perspectives in Modern HPLC: 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: Seven Common Faux Pas in Modern HPLC