Direct determination of glycosylation sites in O-fucosylated glycopeptidesusing nano-electrospray quadrupole time-of-flight mass spectrometry

O-Fucosylation is an unusual posttranslational modification present in seve ral proteins that play important roles in physiological processes such as c oagulation, cell signaling and metastasis. Although the exact function of t he modification is still unclear, the number of proteins found to be modifi ed is increasing, and there is a need for further structural and functional analyses. Here we report on a rapid and straightforward approach in the an alysis of glycosylation status and determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flig ht (nano-ESI Q-TOF) mass spectrometry. In a single measurement of previousl y chemically untreated O-fucosylated peptides originating from the thrombos pondin-l repeats, we were able to determine the glycosylation status of the analyzed peptide, the,glycosylation site, and the glycan structure. The ab undance of glycosylated peptide fragment ions in MS2 spectra suggests that nano-ESI Q-TOF mass spectrometry can be used as a general approach in struc tural studies of O-fucosylation in proteins. Copyright (C) 2001 John Wiley & Sons, Ltd.