We report on the development of a novel methodology to extend the limits of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) general applicability. A sheathless on-line CIE-ESI-MS setup was optimized on standard monosaccharide mixture to operate in reverse polarity and negative ion mode for MS detection without pressure to assist the sample migration, coating of the capillaries, and/or sample derivatization. This approach was further applied for screening of a complex glycopeptide mixture obtained from the urine of a patient diagnosed with N-acetylhexosaminiclase deficiency, known as Schindler's disease. The potential of this methodology in terms of high sensitivity, separation efficiency, resolution, and reproducibility is demonstrated. In combination with the high quality of MS data, a new, significantly improved insight into the sample heterogeneity is possible.