title: Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.
abstract: Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.
authors.affiliation: Swedish Defence Research Agency (FOI), CBRN Defence and Security , SE-901 82 Umeå, Sweden.
authors.forename: Sten-Åke
authors.initials: SÅ
authors.lastname: Fredriksson
authors.forename: Elisabet
authors.initials: E
authors.lastname: Artursson
authors.forename: Tomas
authors.initials: T
authors.lastname: Bergström
authors.forename: Anders
authors.initials: A
authors.lastname: Östin
authors.forename: Calle
authors.initials: C
authors.lastname: Nilsson
authors.forename: Crister
authors.initials: C
authors.lastname: Åstot
mesh_terms: D000036:Abrin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D000328:Adult; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D005690:Galactose / Q000378:metabolism*; D006801:Humans; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D010936:Plant Extracts / Q000737:chemistry*; D054790:Ribosome Inactivating Proteins, Type 2 / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D012276:Ricin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014118:Toxins, Biological / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D010936:Plant Extracts; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C488862:mistletoe lectin I; D000036:Abrin; D012276:Ricin; C049134:volkensin; D014357:Trypsin; D005690:Galactose