Primary structure and glycan moiety characterization of PD-Ss, type 1 ribosome-inactivating proteins from Phytolacca dioica L. seeds, by precursor ion discovery on a Q-TOF mass spectrometer.
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.
Dipartimento di Scienze della Vita, Seconda Università di Napoli, Caserta, Italy.
Angela
A
Chambery
Antimo
A
Di Maro
Augusto
A
Parente
D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D029603:Phytolacca / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D006023:Glycoproteins; D007477:Ions; D010940:Plant Proteins; D011134:Polysaccharides