Instrument response of phosphatidylglycerol lipids with varying fatty acyl chain length in nano-ESI shotgun experiments

In recent years, lipid quantification gained importance. In most cases, this is achieved by spiking the lipid mixture with deuterated standard lipids or lipid analogues that differ in chain length when compared with the natural lipid components. Usually, conventional ESI is employed requiring sample amounts which are not always available. Here, we evaluate the use of nano-ESI for accurate lipid quantification employing deuterated as well as short- and odd-fatty acyl chain analogues. We compare ionisation efficiencies of various phosphatidylglycerol species differing in fatty acyl chain length and saturation. While in our instrumental and experimental set-up differences in ionisation could not be observed for lipids varying in the number of double bonds, short-chain lipid species showed significantly higher intensities when compared with their long-chain analogues. To compensate for these differences and enable accurate quantification using short-fatty acyl chain lipid standards, we generated a calibration curve over a range of lipids with increasing chain length. We tested and evaluated the application of this calibration curve by comparison with a deuterated and odd-chain standard lipid for quantification of lipids in a mixture of known composition as well as a natural lipid extract. The different approaches deliver comparable quantities and are therefore applicable for accurate lipid quantification using nano-ESI. Even though generation of calibration curves might be more laborious, it has the advantage that peak overlap with natural lipids is eliminated and broad peak distributions of deuterated standards do not have to be assessed. Furthermore, it allows the calculation of response factors for long- or short-fatty acyl chain analogues when using deuterated or odd-numbered standard lipids for absolute quantification.