Deamidation in ricin studied by capillary zone electrophoresis- and liquid chromatography–mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2015-01, Vol.974, p.109-117
• Main Authors: Bergström, Tomas, Fredriksson, Sten-Åke, Nilsson, Calle, Åstot, Crister
• Format: Article
• Language: English
• Subjects: Capillarity; Capillary zone electrophoresis; Chromatography; Chromatography, Liquid - methods; Deamidation; Electrophoresis; Electrophoresis, Capillary - methods; In vitro testing; Isoelectric diversity; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Peptides; Plant Extracts - chemistry; Plant Extracts - isolation & purification; Ricin; Ricin - chemistry; Ricin - isolation & purification; Ricinus - chemistry; Toxins
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2014.10.015

•We studied deamidation in ricin toxin from the plant Ricinus communis.•Native/deamidated peptide pairs were analyzed by CZE- and LC–MS.•Three aspargines in ricin were confirmed as being susceptible for deamidation.•The deamidation level in ricin was linked to previously reported isoelectric diversity. 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.