Abstract
Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag](+) and [PC (18:1/16:0) + Ag](+)} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.
Highlights
Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone can have distinct physicochemical properties
To investigate the ability of differential mobility spectrometry (DMS) to separate PC regioisomers, a simple mixture of synthetic lipids comprising 50% PC (16:0/18:1) and 50% PC (18:1/16:0) was prepared and analyzed. This solution was subjected to ESI in positive ion mode to yield ions of m/z 760.4, corresponding to formation of [PC (34:1) + H]+ ions in the gas phase. This ionic form of the PCs was not amenable to DMS separation even when the SV was maximized at 4,100 V and resolving gas was used to increase the ion residence time in the DMS cell
When a solution containing synthetic PC (16:0/18:1) was analyzed under these conditions, two distinct features became visible in the resulting ionogram (Fig. 1). These features were observed at compensation voltage (CV) of 10.7 and 12.3 V when scanning this voltage in the DMS and monitoring product ions from collision-induced dissociation (CID) of m/z 866.5 corresponding to the [PC (16:0/18:1) + Ag]+ cation (Fig. 1A)
Summary
Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. In the absence of rapid and definitive methods for the determination of acyl chain position, assigning sn-position in GPs is often based on the convention of the more unsaturated acyl chain occupying the sn-2 position (see below) This raises concerns that some reported GP structures may be entirely incorrect or ignore the likelihood of both isomers being present in the sample. It has recently been suggested that GP notation be modified to reflect whether the sn-position of the acyl chains has been explicitly determined [22]
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