Hyphenation of Liquid Chromatography and Trapped Ion Mobility - Mass Spectrometry for Characterization of Isomeric Phosphatidylethanolamines with Focus on N -Acylated Species.

In prior research, hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) has demonstrated applicability for characterizing regioisomers in lipidomics studies, including phosphatidylglycerols (PG) and bis(monoacyl)glycerophosphates (BMP). However, there are other lipid regioisomers, such as phosphatidylethanolamines (PE) and lyso- N -acyl-PE (LNAPE), that have not been studied as extensively. Therefore, hyphenated mass spectrometric methods are needed to investigate PE and LNAPE regioisomers individually. The asymmetric structure of LNAPE favors isomeric species, which can result in coelution and chimeric MS/MS spectra. One way to address the challenge of chimeric MS/MS spectra is through mobility-resolved fragmentation using trapped ion mobility spectrometry (TIMS). Therefore, we developed a multidimensional HILIC-TIMS-MS/MS approach for the structural characterization of isomeric phosphatidylethanolamines in both negative and positive ionization modes. The study revealed the complementary fragmentation pattern and ion mobility behavior of LNAPE in both ionization modes, which was confirmed by a self-synthesized LNAPE standard. With this knowledge, a distinction of regioisomeric PE and LNAPE was achieved in human plasma samples. Furthermore, regioisomeric LNAPE species containing at least one unsaturated fatty acid were noted to exhibit a change in collision cross-section in positive ionization mode, leading to a lipid characterization with respect to fatty acyl positional level. Similar mobility behavior was also observed for the biological LNAPE precursor N -acyl-PE (NAPE). Application of this approach to plasma and cereal samples demonstrated its effectiveness in regioisomeric LNAPE and NAPE species' elucidation.