title: Deamidation in ricin studied by capillary zone electrophoresis- and liquid chromatography-mass spectrometry.
abstract: Deamidation in ricin, a toxin present in castor beans from the plant Ricinus communis, was investigated using capillary zone electrophoresis (CZE) and liquid chromatography coupled to high resolution mass spectrometry. Potential sites for deamidation, converting asparagine (Asn) into aspartic or isoaspartic acid (Asp or isoAsp), were identified in silico based on the protein sequence motifs and tertiary structure. In parallel, CZE- and LC-MS-based screening were performed on the digested toxin to detect deamidated peptides. The use of CZE-MS was critical for the separation of small native/deamidated peptide pairs. Selected peptides were subjected to a detailed analysis by tandem mass spectrometry to verify the presence of deamidation and determine its exact position. In the ricin preparation studied, deamidation was confirmed and located to three asparagine residues: Asn54 in the A-chain, and Asn42 and Asn60 in the B-chain. Possible in vitro deamidation occurring during sample preparation was monitored using a synthetic peptide with a known and rapid rate of deamidation. Finally, we showed that the isoelectric diversity previously reported in ricin is related to the level of deamidation.
authors.affiliation: Swedish Defence Research Agency, CBRN Defence and Security, Cementvägen 20, SE-901 82 Umeå, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Bergström
authors.affiliation: Swedish Defence Research Agency, CBRN Defence and Security, Cementvägen 20, SE-901 82 Umeå, Sweden.
authors.forename: Sten-Åke
authors.initials: SÅ
authors.lastname: Fredriksson
authors.affiliation: Swedish Defence Research Agency, CBRN Defence and Security, Cementvägen 20, SE-901 82 Umeå, Sweden.
authors.forename: Calle
authors.initials: C
authors.lastname: Nilsson
authors.affiliation: Swedish Defence Research Agency, CBRN Defence and Security, Cementvägen 20, SE-901 82 Umeå, Sweden. Electronic address: astot@foi.se.
authors.forename: Crister
authors.initials: C
authors.lastname: Åstot
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010936:Plant Extracts / Q000737:chemistry* / Q000302:isolation & purification; D012276:Ricin / Q000737:chemistry* / Q000302:isolation & purification; D012278:Ricinus / Q000737:chemistry*
chemical_list: D010936:Plant Extracts; D012276:Ricin
keywords: Capillary zone electrophoresis; Deamidation; Isoelectric diversity; Liquid chromatography; Mass spectrometry; Ricin