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.T ype 2 ribosome-inactivating proteins (RIP-II toxins) are a class of plant toxins that includes a number of potent chem-bio threat agents, such as ricin (Ricinus communis), abrin (Abrus precatorius), viscumin (Viscum album), modeccin (Adenia digitata), and volkensin (Adenia volkensii). RIP-II toxins are heterodimeric proteins that consist of an enzymatically active N-glycosidase A chain connected to a B chain via a disulfide bond. The B chain is a lectin with carbohydrate binding domains that have an affinity for galactose-terminated surface receptors on eukaryotic cells. Binding promotes the uptake of the toxin into the cell by endocytosis, where a part intriguingly finds its way via the Golgi complex to the endoplasmatic reticulum. From there the A subunit is translocated into the cytosol, where it hydrolyses the Nglycosidic bond between an adenine residue and ribose at a specific position in 28S rRNA and thereby inhibits protein synthesis, eventually causing cell death.

Comparison of microbial and transient expression (tobacco plants and plant‐cell packs) for the production and purification of the anticancer mistletoe lectin viscumin

“…Second, the linker sequence between the A and B chains can trigger Golgi‐mediated transport (Hillmer, Movafeghi, Robinson, & Hinz, ) of the glycosylated pro‐toxin from the ER to storage vacuoles (Frigerio et al, ), where the linker is removed by a vacuolar endoproteinase (Lord, ) and the active toxin is safely stored. These beneficial effects of the linker are a common feature of type II RIPs (Fredriksson et al, ) and would clearly not be expected when coexpressing the individual chains (visA + visB). Accordingly, we observed a gradient of deleterious effects (browning, low TSP, and low product accumulation) with visA showing the greatest severity, followed by visA + visB and finally visFL.…”

LC-HRMS Screening and Identification of Novel Peptide Markers of Ricin Based on Multiple Protease Digestion Strategies

“…Peptide markers of ricin from trypsin digestion have been mainly produced by digestion after denaturation, reduction and alkylation [14,21], or direct digestion of intact ricin [13,18]. To fully understand the profile of tryptic peptide markers of ricin, efforts were made to screen them by digestion under both reduction and non-reduction.…”

“…The unique intra-disulfide linked peptides of both T3B-ss-T5B and T14B-ss-T16B were identified. In addition, one common inter-disulfide T24A-ss-T1B between intact ricin and RCA120 was also detected, which has been considered as an important peptide marker for the presence of intact ricin or RCA120 [18].…”

“…Some efforts were made to enhance efficiency of trypsin digestion. For example, instead of trypsin digestion following the denaturation, reduction and alkylation, the direct trypsin digestion of intact ricin under the solvent-assisted condition was performed with no missed cleavage and reduced digestion time [13,18]. Besides, two peptide markers (C18A and C18B) generated from chymotrypsin digestion were also applied for unambiguous identification of ricin from crude extracts of castor bean and soil samples [19].…”

Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples

“…Another similar method includes a shortened digestion time without prior reduction of disulfide bonds, in which the peptides from the A-chain, peptides from the B-chain and the peptide containing the intact interchain disulfide bond verifying the presence of the link between the A- and B-chains were identified and differentiated from RCA120 [ 25 ]. This approach was reported for the identification of ricin spiked into complex matrices such as milk, apple juice, serum and saliva [ 26 ], in surface swabs from a public health investigation [ 27 ], as well as identification of ricin and abrin in a forensic investigation [ 28 ].…”