Figure 3. ESI-MS and ESI-MS/MS of TbFUT1 in vitro reaction product.

(A) ESI-MS of the purified and permethylated reaction product. The ion at m/z 692.5 is consistent with the [M + Na]⁺ ion of a permethylated trisaccharide of composition dHex₁Hex₁HexNAc₁. Some of the unmodified acceptor (Hex₁HexNAc₁) was also observed (m/z 518.4). (B) MS/MS product ion spectrum of m/z 692.5. The collision-induced fragmentation pattern indicated that the dHex (Fuc) residue was linked to the Hex (Gal) and not to the HexNAc (GlcNAc) residue.

Figure 3—figure supplement 1. Preliminary characterization of TbFUT1 reaction products.

(A) Labelled products were recovered from the activity assays and treated with 4 M trifluoroacetic acid (TFA) (acid hydrolysis). (B) Tritiated products, recovered from the TbFUT1 activity assays, were treated (lanes 1 and 3) with 10 U of X. manihotis α-1,2-fucosidase or mock treated (lanes 2 and 4) by incubating with only the fucosidase buffer. Acid hydrolysis reactions, fucosidase treatments, and monosaccharide standards were run on a HPTLC plate in solvent A. The radiolabelled products were visualized by fluorography, and the standards were visualized with orcinol/sulphuric acid (C).

Figure 3—figure supplement 2. Purification of the TbFUT1 reaction product by normal phase HPLC.

An aliquot (2%) from each sugar-containing fraction from the normal phase purification was run on a HPTLC plate in solvent A and developed with orcinol/sulphuric acid. Standards for the acceptor (LNB) and for Fuc were also analysed. Fractions containing the reaction product (33 and 34) were pooled for analysis.