Figure 4.

Determination of apparent kinetic parameters for Δ56ST6Gal I and Δ56ST3Gal I towards the unnatural donor substrate CMP‐SiaNAl. Incubations in 6.5 μL of culture medium of transfected HEK293 cells containing recombinant Δ56ST3Gal I or Δ56ST6Gal I (or no enzyme, mock) were carried out for 6 h with various concentrations of CMP‐SiaNAl (donor substrate) and 200 μg of asialofetuin. Sialic acids from each reaction were released, derivatized with DMB and subjected to LC‐MS³ analysis to quantify SiaNAl. A) The background signal (mock) was subtracted, and data were fitted to the Michaelis–Menten equation; apparent kinetic parameters for Δ56ST6Gal I and Δ56ST3Gal I were calculated for CMP‐SiaNAl (Table 2). B) Quantification was based on LC‐MS signal intensity from MRM‐MS³ analysis of the DMB‐SiaNAl specific [M+H]⁺ product at m/z 446. C) MS3 fragmentation pattern of [M+H]⁺ ion at m/z 446; D) Major fragmentation products.