Characterization of the Forssman Glycolipid Hapten of Horse Kidney by Mass Spectrometry

The Forssman glycolipid hapten was isolated from horse kidney and analyzed by direct inlet mass spectrometry of undergraded lipid derivatives. Methylated glycolipid gave no molecular ions, but some important carbohydrate sequence and ceramide (fatty acid and long chain base) ions. After reduction of this derivative with LiAlH4 (amide group of ceramide and of amino sugars were converted to the corresponding amines) a molecular weight ion (at m/e 1782) was recorded, as well as ions which were conclusive for the type and exact ratio of sugars and their sequence. Without any conventional degradative studies it was possible to conclude that the glycolipid was a pentaglycosyl-ceramide with the sequence hexosamine-hexosamine-hexose-hexose-hexose-ceramide, as first proposed by B. Siddiqui and S. Hakomori ((1971) J. Biol. Chem. 246, 5766–5769). The fatty acids were mainly nervonic (24:1) and lignoceric (24:0) acid but also C16 to C23 fatty acids. Both sphingosine and phytosphingosine were present. Evidence was obtained from the spectra for a preferential combination of long chain (24) fatty acid and phytosphingosine.