Structural Characterization of a Novel Tetrasaccharide Attached to Ser-61 of Human Factor IX by Mass Spectrometry and 1H NMR Spectroscopy

Publisher Summary This chapter explores the structural characterization of a novel tetrasaccharide attached to ser-61 of human factor ix by mass spectrometry and 1 H NMR spectroscopy. In a study described in the chapter, trypsin-digested S-carboxymethylated human plasma factor IX was prepared. No mass was observed that corresponded to any of the expected forms of the factor IX 44-80 tryptic peptide. The 44-80 peptide sequence is QYVDGDQCE S NPCLNGG S CK D DINSYECWCPFGFEGK. Tryptic RP-HPLC fractions were then subjected to amino acid analysis to identify a doublet peak containing the 44–80 peptides. The mass obtained for this fraction indicated the presence of some unknown modification that added 801.2 amu to the expected glycopeptide mass, far in excess of the 146.2 amu indicative of O -linked fucose. Digestion of the 44–80 peptides with thermolysin produced fragments demonstrating that the unknown -802 amu modification was found within residues 57–65. Digestion with α-fucosidase had no effect on the 57–65 glycopeptide, and the ES–MS fragmentation pattern indicated that fucose was present at the reducing end.