Fig. 4
Cleavage position when digesting (a) D4 and (b) D5 with CSN, CSN-Y270A, CSN-E59A, and CSN-W118A. The enzymes were incubated with 1 mM of 18O-labeled D4 or D5 at 30 °C for 10 min and the products were immediately measured using UHPLC-ELSD-ESI-MS1. The enzyme concentrations when using D4 were 0.02 μM for CSN and 0.035 μM for CSN-Y270A. When using D5, 0.16 μM of CSN, 0.42 μM of CSN-Y270A, 0.33 μM of CSN-E59A, and 5 μM of CSN-W118A were applied. Quantification of the oligomers with and without the label at the reducing end was done by comparison with external oligomer standards. The amounts of D1 (faded bars) were not determined by quantification of D1 but instead deduced from the measured amounts of D3 or D4. The combined amount of all oligomers was set to 1 for each of the enzymes. Three independent enzyme batches were used for each enzyme, and the experiments were performed as triplicates for each batch. Data given are the mean values of all nine replicates and the standard deviations between the three independent enzyme batches are indicated