High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm

Abstract

The choline head group containing phosphatidylcholine (PC) and sphingomyelin (SPM) are major eukaryotic lipid components playing an important role in forming membrane microdomains and serve as precursor of signaling molecules. Both lipids can be monitored by positive ion mode electrospray tandem mass spectrometry using a parent ion scan of m/z 184. Although PC species appear at even m/z and SPM species at odd m/z, there may be a significant overlap of their isotopes. In order to separate PC and SPM species, an isotope correction algorithm was established, which utilizes calculated isotope percentages to correct the measured peak intensities for their isotopic overlap. We could demonstrate that this approach was applicable to correct the isotope overlap resulting from spiked PC and SPM species. Quantification was achieved by addition of different PC and SPM species prior to lipid extraction. The developed assay showed a precision, detection limit and robustness sufficient for routine analysis. Furthermore, an analysis time of only 1.3 min combined with automated data analysis using self-programmed Excel Macros allows high-throughput analysis. In summary, this assay may be a valuable tool for detailed lipid analysis of PC and SPM species in a variety of sample materials.

Introduction

A major component of eukaryotic lipid membranes are phospholipids, including the glycerol-based phosphatidylcholine (PC) and the sphingosine-based sphingomyelin (SPM) [1]. These lipids vary in the degree of saturation and chain length of the fatty acid moiety. In addition, there exists a minor PC fraction containing an ether bond at sn-1 position instead of the ester bond [2], which may serve as precursor for the generation of platelet-activating factor (PAF), a potent lipid mediator [3]. These structural differences may have a great influence on membrane properties, e.g., by modulating the formation of lipid raft structures [4]. Thus, Triton X-100 detergent-insoluble lipid microdomains are enriched in saturated PC species compared to the soluble membrane fraction [5]. PC and SPM also serve as precursors for a variety of signaling molecules including lysophosphatidylcholine [6], diacylglycerol [7] and ceramide [8] resulting from agonist induced action of different lipases [9]. Additionally, SPM exhibits a tight interaction with cholesterol, which may influence cellular cholesterol homeostasis [10] as well as lipoprotein function. Thus, the lecithin-cholesterol acyltransferase (LCAT) reaction in reconstituted high density lipoproteins is inhibited by addition of SPM [11]. Moreover, an increased SPM to PC ratio enhances the susceptibility of low density lipoproteins (LDL) to secretory sphingomyelinase [12]. Sphingomyelinase mediated ceramide generation leads to the formation of aggregated LDL, which exhibits a high atherogenic potential. In summary, SPM and PC are involved in the regulation of several cellular processes as well as in the modulation of lipoprotein function, which may be of special pathophysiological relevance.

A large number of methodologies have been described for quantitative phospholipid analysis including high performance liquid chromatography [13] and thin layer chromatography [14], [15], [16]. However, these conventional methods usually do not provide information concerning the fatty acid chains. Additionally, these methods often are time-consuming multistep procedures and lack sensitivity essential for the analysis of cellular subfractions. In the last years numerous mass spectrometric approaches with different ionization techniques were used to characterize and quantify phospholipid species [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29]. The most commonly used technique is electrospray ionization tandem mass spectrometry (ESI-MS/MS) allowing the quantification of different phospholipid classes from crude lipid extracts [30]. In positive ion mode, both PC and SPM are monitored by the collision induced formation of a m/z 184 fragment specific for the phosphocholine head group, whereas in negative ion mode only SPM is detected by a fragment of m/z 168 resulting from an in source demethylation of the phosphocholine head group [30]. However, SPM analysis in negative ion mode exhibits a reduced sensitivity compared to positive ion mode measurement [30]. In positive ion mode the protonated PC species appear at even m/z and SPM species at odd m/z values. In principle, the different m/z allow a discrimination of these lipid classes, but frequently unseparated lipid extracts reveal a relevant overlap of SPM and PC isotope peaks. The aim of this study was to develop a high-throughput ESI-MS/MS method using direct flow injection of crude lipid extracts, which allows a separate quantification of PC and SPM by correction of the overlapping isotope peaks.