Extended Data Fig. 7: Enzymatic extension of 4-mer and 5-mer acceptor primers with single sugar residues.

To complement the UDP-Glo assays performed in Fig. 4 and Extended Data Fig. 5, we analyzed the reaction products for glycan extension by MALDI-MS. Individual spectra were obtained for the indicated enzyme, sugar nucleotide donors (a and b), and the 4-mer-pNP (c) and 5-mer-pNP (e) acceptors. Electrospray mass spectra of each of the synthetic acceptors indicated a predicted parent mass of 897.75 and 1073.87 for the 4-mer-pNP and 5-mer-pNP acceptors, respectively (c and f). However, MALDI-MS of the compounds produced a broad pair of mass peaks where the smaller, higher mass peak matched the predicted mass (singly charged masses of 898 and 1074 for the 4-mer-pNP and 5-mer-pNP acceptor, respectively, insets for panels c and f at the top). For each compound, the more abundant second species was 15 mass units smaller than the predicted mass, consistent with an in-source rearrangement resulting in neutral mass loss during MALDI analysis. The cause of this 15 mass unit neutral loss is not clear at the present time, but it did not impact the ability of the 4-mer and 5-mer to act as acceptors for extension by EXT1-2. Reactions containing the 4-mer acceptor, which harbored a reducing terminal GlcNAc residue, could be extended by the mass of a GlcA unit in the presence of the UDP-GlcA donor (e), while reactions containing the UDP-GlcNAc donor led to no extension (d). By comparison, reactions containing the 5-mer acceptor (harboring a reducing terminal GlcA residue) could be extended by the mass of a GlcNAc residue in the presence of a UDP-GlcNAc donor (g), but reactions containing UDP-GlcA as donor led to no extension (h). Data presented are representative of n > 3 independent reactions.