Linear ion-trap MSⁿ with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis

1-O-acylceramide is a new class of epidermal ceramide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSⁿ) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]⁺ ions were subjected to LIT MS² to yield predominately the [M + H-H₂O]⁺ ions, followed by MS³ to cleave the 1-O-acyl residue to yield the [M + H-H₂O-(1-O-FA)]⁺ ions. The structures of the N-acyl chain and longchain base (LCB) of the molecule were determined by MS4 on [M + H-H₂O-(1-O-FA)]⁺ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0-to 30:0-O-acyl) and N-acyl chains (from 14:0-to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N-or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family.