Quench-flow analysis

To resolve rapid single-encounter events for USP14-catalyzed deubiquitination, millisecond reactions were performed on a quench-flow device (Kintek RQF-3). The quench-flow machine was first calibrated with known amount of BSA, ovalbumin, and UbcH10 to achieve maximum recovery of samples according to the manufacturer’s instructions. Generally, the recovery ranged between 75 to 85% of the input and was relatively even among the reaction loops (3.5 milliseconds to minutes). For these reactions, human proteasomes were purified on a large scale as described above and concentrated to 2 μM or higher in buffer M (50 mM Tris-HCl [pH 7.5], 1 mM MgCl₂, 1 mM ATP, and 5% glycerol). Recombinant USP14 was purified and concentrated to 250 μM or higher in buffer N (35 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM DTT, and 5% glycerol). Before the quench-flow reaction, proteasomes were pre-incubated with USP14, and the solution was adjusted to 50 mM Tris-HCl [pH 7.5], 3.5 mM NaCl, 3 mM MgCl₂, 3 mM ATP, 0.74 mM DTT, and 3.3% glycerol. For the proteasome-only control (i.e., no USP14), only the buffer N was added as a mock to achieve the same final composition of buffer ingredients as above. Ubiquitinated NCB1 was generated on a large scale, and before the reaction, adjusted to about 0.47 μM in buffer O (20 mM Tris-HCl [pH 7.5], 36 mM KCl, 3 mM ATP, and 3 mM MgCl₂). To enhance the recovery, prevent aggregation, and reduce non-specific binding to the tubing, 1 mg/ml of BSA and 0.0025% of NP-40 (each final concentration after mixing with enzymes) were added to the conjugate solution. Ubiquitinated full length cyclin B1 was essentially prepared as above except adjusting the substrate at ~0.17 μM.

Millisecond single-encounter reactions were carried out from 5 milliseconds to minutes by mixing the two reactants in the corresponding reaction loops and then quenched in the device with 3.3x quench solution P (300 mM Tris-HCl [pH 6.8], 3% SDS, and 10% glycerol) with freshly added 330 mM DTT. A small amount of SDS-PAGE loading dye was then added to each collected sample. For the 0 sec sample, enzymes or ubiquitin conjugates alone were separately quenched inside the machine with each corresponding mock buffer by utilizing reaction loop 1 at 3.5 msec. The reactions were resolved in SDS-PAGE/immunoblotting.

For quantification, the total ubiquitin conjugate or each conjugate species was analysed by Amersham 600 RGB system (GE Healthcare) or Odyssey CLx Infrared Imaging System (LI-COR Biosciences). The conjugate signal was normalized by UbcH10 for each time point to correct for recovery, and then further normalized to the 0 time control. For Ub_n-NCB1, proteasome alone showed some non-specific signals detected by HA antibody. This number was subtracted before normalization. The data were curve-fitted by non-linear regression with the best fit model using GraphPad Prism (GraphPad Software).