LC-MS Meets MS Imaging: Using CCS for Confident Lipid Annotation

The involvement of lipids in many biological and physiological processes makes their analysis of paramount importance. High-resolution mass spectrometry (HR-MS) combined with fragmentation experiments can identify lipid species. However, to differentiate isomeric lipids or isobaric interferences, powerful additional separation techniques, often (ultra)high performance liquid chromatography, are required.

Trapped ion mobility-mass spectrometry (TIMS) for the separation of ions after matrix-assisted laser desorption/ionization (MALDI) has become a useful tool in lipid research, increasing the reliability of lipid identification using complementary collision cross section (CCS) data.

In this study, we created a tissue-specific CCS database using hydrophilic interaction liquid chromatography coupled to electrospray TIMS-MS. With the help of this database, we were able to distinguish isomeric lipid classes clearly, such as phosphatidylglycerol (PG) and its regio-isomer bis(monoacylgylcero)phosphate (BMP), as well as isobaric interferences due to adduct formation during the MALDI process. The developed workflow and the application to a mouse spleen sample for confident lipid identification applying MALDI-TIMS-MS imaging will be presented.

• Use trapped ion mobility separation to identify isomeric lipids confidently
• Separate BMP and PG lipids using TIMS
• Use CCS to visualize confident lipid annotations spatially on tissue
• Build tissue-specific CCS databases