Structural Elucidation of Phosphatidylcholines via Radical-Directed Dissociation of [M + Cu ^II + Anion] ⁺ Ion Types

Tandem mass spectrometry (MS/MS) is frequently used in phospholipid characterization to determine lipid class, fatty acyl chain length, and degree of unsaturation. However, conventional MS/MS methods are limited in characterizing isomeric lipids resulting from variants in double bond and <i>sn</i>-positions within the fatty acyl chains. This limitation is due to the fact that conventional collision induced dissociation (CID) of even-electron lipid precursor ions does not generate highly efficient product ions from intrachain fragmentation of the fatty acyl substituents. Herein, we have developed a workflow that leverages a new lipid ion type to facilitate radical-directed dissociation (RDD). Briefly, low-energy CID of [M + Cu^II + anion]⁺ ion types results in a radical cation, and subsequent activation of the radical cation results in highly efficient intrachain fragmentation of fatty acyl chains. This method provides abundant diagnostic fragment ions that are associated with the double bond and fatty acyl chain positions on the glycerol backbone and thus can be used to differentiate isomeric phosphatidylcholines (PCs). The incorporation of an anionic ligand in the [PC + Cu^II + anion]⁺ ion type is key to this chemistry. Specifically, the major fragmentation channel for the [PC + Cu^I]⁺ ion type is neutral loss of phosphocholine headgroup but shifts to RDD for [PC + Cu^II + anion]⁺ ion types containing strongly electronegative anions.