Abstract
Diacyl glycerophospholipids (GPs) belong to the most abundant lipid species in living organisms and consist of a glycerol backbone with fatty acyl groups in sn-1 and sn-2 and a polar head group in the sn-3 position. Regioisomeric mixed diacyl GPs have the same fatty acyl composition but differ in their allocation to sn-1 or sn-2 of the glycerol unit. In-depth analysis of regioisomeric mixed diacyl GP species composed of fatty acyl moieties that are similar in length and degree of saturation typically requires either chemical derivatization or sophisticated analytical instrumentation, since these types of regioisomers are not well resolved under standard ultra-performance liquid chromatography (UPLC) conditions. Here, we introduce a simple and fast method for diacyl GP regioisomer analysis employing UPLC tandem mass spectrometry (MS/MS). This GP regioisomer analysis is based both on minor chromatographic retention time shifts and on major differences in relative abundances of the two fatty acyl anion fragments observed in MS/MS. To monitor these differences with optimal precision, MS/MS spectra are recorded continuously over the UPLC elution profile of the lipid species of interest. Quantification of relative abundances of the regioisomers was performed by algorithms that we have developed for this purpose. The method was applied to commercially available mixed diacyl GP standards and to total lipid extracts of Escherichia coli (E. coli) and bovine liver. To validate our results, we determined regioisomeric ratios of phosphatidylcholine (PC) standards using phospholipase A2-specific release of fatty acids from the sn-2 position of the glycerol backbone. Our results show that most analyzed mixed diacyl GPs of biological origin exhibit significantly higher regioisomeric purity than synthetic lipid standards. In summary, this method can be implemented in routine LC-MS/MS-based lipidomics workflows without the necessity for additional chemical additives, derivatizations, or instrumentation.
Highlights
Glycerophospholipids (GPs) are the ubiquitous building blocks of biological membranes in bacteria and eukaryotes.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Different types and compositions of head groups and hydrocarbon chain(s) are the basis for the multitude of distinct GP classes and species, which contribute to the complexity of biological membranes.In recent years, mass spectrometry–based lipidomics has made substantial progress in quantitative analysis of lipids [1]
Several hundreds of molecular lipid species can be identified in a single LC-MS/MS run by analyzing combined molecular weight and fragment ion data [2]
Based on targeted MS/MS combined with the new types of graphic and numeric data evaluation introduced here, we demonstrate the suitability of regular UPLCMS/MS as a solitary technique for determining the regioisomeric composition of GP standards and of GPs of biological origin
Summary
Glycerophospholipids (GPs) are the ubiquitous building blocks of biological membranes in bacteria and eukaryotes. This feature has been used to establish calibration curves, correlating the composition of PC regioisomer mixtures to the sn-2/sn-1 fatty acid anion fragment intensity ratio [12] This concept, ideally requires the availability of a pure regioisomeric standard for each species. A recent report describes selective detection of GP regioisomers by a hybrid MS3 approach It employs CID followed by ultraviolet photodissociation to determine sn positions of fatty acyl chains via position-specific product ions [16]. The most recent methods employ complex and advanced mass spectrometric instrumentation allowing regioisomer recognition by, e.g., specific gas-phase chemical reactions or laser-induced fragmentations These advanced techniques generate regioisomer-specific fragment ions or achieve complete regioisomer separation before their analysis by MS/MS; the essential instrumentation is commonly not available in MS facilities. Based on targeted MS/MS combined with the new types of graphic and numeric data evaluation introduced here, we demonstrate the suitability of regular UPLCMS/MS as a solitary technique for determining the regioisomeric composition of GP standards and of GPs of biological origin
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