Seeds from Phytolacca dioica L. contain at least three N-glycosylated PD-Ss, type 1 ribosome-inactivating proteins (RIPs), which were separated and purified to homogeneity by conventional chromatographic techniques. ESI-Q-TOF mass spectrometry provided the accurate M(r) of native PD-S1 and PD-S3 (30957.1 and 29785.1, respectively) and the major form PD-S2 (30753.8). As the amino acid sequence of PD-S2 was already known, its disulfide pairing was determined and found to be Cys34-Cys262 and Cys88-Cys110. Further structural characterization of PD-S1 and PD-S3 (N-terminal sequence determination up to residue 30, amino acid analysis and tryptic peptide mapping) showed that the three PD-Ss shared the entire protein sequence. To explain the different chromatographic behaviour, their glycosylation patterns were characterized by a fast and sensitive mass spectrometry-based approach, applying a precursor ion discovery mode on a Q-TOF mass spectrometer. A standard plant paucidomannosidic N-glycosylation pattern [Hex(3), HexNAc(2), deoxyhexose(1), pentose(1)] was found for PD-S1 and PD-S2 on Asn120. Furthermore, a glycosylation site carrying only a HexNAc residue was identified on Asn112 in PD-S1 and PD-S3. Finally, considering the two disulfide bridges and the glycan moieties, the experimental M(r) values were in agreement with the mass values calculated from the primary structure. The complete characterization of PD-Ss shows the high potential of mass spectrometry to rapidly characterize proteins, widespread in eukaryotes, differing only in their glycosylation motifs.