Structural identification of novel bound 1-O-acylceramides in human stratum corneum and murine epidermis using mass spectrometry

Abstract The stratum corneum (SC)—the outermost layer of the skin—serves as a barrier that protects the body from environmental stress and prevents transdermal moisture loss. Skin ceramides comprise a sphingoid base backbone linked via an amide bond to a fatty acid. Fatty acids in ceramides are classified into four types: non-hydroxy (N), α-hydroxy (A), ω-hydroxy (O), and esterified ω-hydroxy (EO). Among these, O-type ceramides are ester-linked via their ω-hydroxyl group to the cornified envelope protein. While O-type ceramides containing four distinct sphingoid bases have been reported, bound-type acylceramides featuring an additional acyl group esterified to the sphingoid hydroxyl remain unreported. Therefore, this study aims to identify and characterize 1- O -esterified O-type phytoceramide (1- O- EOP) as a novel class of bound ceramides in human SC and murine epidermis. Using high-resolution mass spectrometry and direct infusion-ion trap mass spectrometry, we show that these ceramides are esterified with an additional fatty acid at the 1-hydroxy group of the phytosphingosine backbone within ω-hydroxy ceramides. Based on the tandem mass spectrometry fragmentation patterns of 1- O -EOP ceramides, comprehensive fragmentation schemes are proposed. In addition, we have developed a method to identify and profile 1- O -EOP ceramides in human SC and murine epidermis, providing a foundation for detecting bound 1- O -acylceramides in mammalian skin.