Separation and mass spectrometric characterization of covalently bound skin ceramides using LC/APCI-MS and Nano-ESI-MS/MS.

Ceramides covalently bound to keratinocytes are essential for the barrier function of the skin, which can be disturbed in diseases, such as psoriasis and atopic dermatitis. These ceramides of the classes omega-hydroxyacyl-sphingosine and omega-hydroxyacyl-6-hydroxysphingosine contain an omega-hydroxy fatty acid. For their separation and identification, a new analytical approach based on normal phase liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry and tandem nano-electrospray mass spectrometry, respectively, is presented here.

Site-specific cleavage--a model system for the identification of lipid-modified glutamate residues in proteins.

Numerous proteins are modified post-translationally after their biosynthesis at the ribosomes of the cell. One such modification, only poorly characterized to date, is the formation of lipid esters of glutamate side chains in the skin proteins of land-living mammals; here a subset of very long chain fatty acids, ceramides and/or glucosylceramides, are bound through their omega-hydroxy groups to structural proteins of the so-called "cornified envelope" in the outermost layer of the skin, the stratum corneum. We report an approach for the identification of proteins containing ester-modified glutamic acid residues and the determination of their positions within the peptide sequence, designed for mass spectrometric investigation of human skin proteins.

Sphingolipid activator proteins: proteins with complex functions in lipid degradation and skin biogenesis.

Sphingolipid activator proteins (SAPs or saposins) are essential cofactors for the lysosomal degradation of membrane-anchored sphingolipids. Four of the five known proteins of this class, SAPs A--D, derive from a single precursor protein and show high homology, whereas the fifth protein, GM2AP, is larger and displays a different secondary structure. Although the main function of all five proteins is assumed to lie in the activation of lipid degradation, their specificities and modes of action seem to differ considerably.