An analytical approach based on sheathless on-line coupling of capillary electrophoresis (CE) and electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been developed for providing new insight into the characterization of carbohydrate mixtures. The home-built sheathless CE/ESI microspray interface consisting of an one-piece CE column has been optimized and applied for the screening of a complex mixture of O-glycosylated peptides obtained and previously purified from urine of patients suffering from N-acetylhexosaminidase deficiency. The separation efficiency and the resolution obtained in CE/UV experiments could be reproduced with high sensitivity in on-line CE/MS runs under mild ES-Inegative ion mode conditions, due to the compatibility of the reconsidered CE-QTOF/MS operating parameters, microspray tip performance and the use of MS friendly volatile CE buffer system as ammonium acetate/ammonium hydroxide. By employing the high speed automated "on-the-fly" MS-MS/MS switching abilities of QTOF/MS instrument, the multicomponent sample could be separated and subsequently submitted to both MS and low-energy collision-induced dissociation of selected precursor ions. Using the on-line CE-ESI-MS/MS coupling, glycosylated sialylated peptides undetectable in complex mixtures by a direct ESI/MS analysis could be identified.