LOCALIZATION OF O-GLYCOSYLATION SITES OF MUC1 TANDEM REPEATS BY QTOF ESI MASS-SPECTROMETRY

The potential of electrospray mass spectrometry (ESMS) for the sequenc ing of glycopeptides was evaluated using quadrupole time-of-flight (QT OF) technology in the MS/MS mode. The location of O-glycosylation site s was possible in the positive ion (+) mode by detection of prominent y-and b-fragment ions from the underivatized TAP25-2 [T(1)APPAHGVT(9)S (10)APDT(14)RPAPGS(20)T(21)APPA], an overlapping sequence of MUC1 tand em repeats which had been glycosylated in vitro by two GalNAc residues in the positions T9 and T21, The high mass resolution and accuracy of QTOF-(+)ESMS allowed reliable structural assignments. The reduced com plexity of the fragment spectra and the higher signal-to-noise ratio r ender QTOF-(+)ESMS an alternative mass spectrometric approach to the i dentification of O-glycosylation sites when compared with sequencing b y post-source decay matrix-assisted laser desorption/ionization MS. Di agnostic ions from the N-terminus in the b-series offered direct evide nce, which was supported by indirect evidence from the C-terminus ions of the y-series, The higher glycosylated GalNAc(2)-substituted fragme nts were mainly observed as multiply ionized species. (C) 1998 John Wi ley & Sons, Ltd.