N-Glycosylation Profile of Abrin Certified EU Reference Material

Abrin is a highly toxic plant protein encompassing four isoforms, abrin-a, -b, -c and -d. An abrin reference material was isolated from <i>Abrus precatorius</i> and certified (EURM-113) by the EuroBioTox consortium. Here, we present a detailed characterisation of the <i>N</i>-glycosylation profile of EURM-113. The monosaccharide composition of the <i>N</i>-glycans was determined and quantified. Release of the <i>N</i>-glycans yielded 13 different partially xylosylated, oligomannosidic and paucimannosidic glycan structures. Two <i>N</i>-glycans were found at N82 and N110 of the abrin-b A-chain and another two at N100 and N140 of the B-chains. The <i>N</i>-glycosylation sites N200 in the A-chain and N141 in the B-chain were non-glycosylated. Whereas N82 and N110 of abrin-b comprised paucimannosidic glycans, N100 and N140 of the B-chains revealed oligomannosidic <i>N</i>-glycans. Xylose was absent in the glycans at N100 but was present in about half of the glycans at N140. Hence, this study revealed substantially different types of glycan structures within the B-chains compared to the abrin-b A-chain. Furthermore, the most C-terminal <i>N</i>-glycosylation site in the A-chain was found to be non-glycosylated in all abrin isoforms detected. Additionally, the establishment of the <i>N</i>-glycosylation profile of the abrin reference material led to the identification of the abrin isoforms -a, -b and -c. In conclusion, the abrin <i>N</i>-glycosylation profile is highly similar to the one of ricin and yields high analytical value to be further exploited as a fingerprint in forensic investigations to uncover toxin production or toxin provenance.