Figure 4.

nMS method testing PROTAC cooperativity. Top: VCB (5 μM) + AT1 (10 μM). Middle: substrate protein (5 μM) + AT1 (10 μM). Bottom: VCB (5 μM) + AT1 (10 μM) + substrate protein (5 μM). (a) PROTAC cooperativity with Brd4^BD2 as substrate protein. (b) PROTAC cooperativity with Brd4^BD1 as substrate protein. The insets show the estimated fraction of integrated peaks corresponding to the labeled species. For Brd4^BD2, only the peaks which correspond to [M + 6H]⁶⁺ and [M + 7H]⁷⁺ are used for the quantification and, for Brd4^BD1, only [M + 5H]⁵⁺, [M + 6H]⁶⁺, and [M + 7H]⁷⁺. Top and middle panels correspond to single measurements, while those in the bottom panel are the average of three measurements, with error bars representing standard deviations. Values for α (top) are taken from Roy et al.¹⁶