Evaluation of Lipid In-Source Fragmentation on Different Orbitrap-based Mass Spectrometers

Natural lipidomes represent a complex mixture of lipid molecular species with a variety of biological and signaling functions. Modern mass spectrometry (MS)-based analytical platforms are often used to resolve the complexity of natural lipidomes. The quantitative transfer of lipid molecular species in the gas phase during the electrospray ionization required for MS analysis might be challenged by lipid in-source fragmentation (ISF) hampering their accurate identification and quantification. Here we evaluated the effect of transmission radio frequency (RF) levels and ion transfer temperatures (ITTs) on the analysis of four different lipids (ceramide, cholesteryl ester, phosphatidylethanolamine, and triacylglyceride) ionized in positive ion mode on three different Orbitrap-based platforms. ITT and RF levels were ramped in a systematic way to determine the best settings, allowing the most sensitive detection accompanied by the lowest ISF of a lipid. The extent of the ISF was shown to depend on the configurations of the transmission devices (S-lens vs letterbox/ion funnel) at defined RF and ITT levels for each studied lipid class. We provide here the recommendations for reducing the extent of lipid ISF without a significant loss in sensitivity for Q Exactive HF, Q Exactive HF-X, and Orbitrap Fusion Lumos platforms.