HPLC Separation of PapB and QueE Variants

All of the assays and controls were analyzed as described (30 (link), 40 (link)) with the following modifications for the assays that contained both prereduced wildtype (WT) PapB and QueE. A 50-μl aliquot was injected onto a Hypersil GOLD C18 column (2.1 mm x 150 mm, 1.9 mm particle size) (Thermo Fisher) preequilibrated in 0.1% (v/v) Optima TFA (Fisher) in LC-MS Optima water (Fisher). The separation consisted of washing with 100% A (0.1% [v/v] TFA in Optima water) from 0 to 3 min, followed by a linear gradient from 0 to 40% B (0.1% [v/v] TFA in Optima acetonitrile) from 3 to 20 min, followed by a linear gradient from 40% B to 100% B from 20 to 23 min, washing with 100% B from 23 to 26.5 min, and re-equilibration into 100% A from 26.5 to 30.1 min.
The assays that contained a PapB variant and QueE were analyzed as follows. A 20-μl aliquot was injected onto a Hypersil GOLD C18 column (2.1 mm x 150 mm, 1.9 mm particle size) (Thermo Fisher) preequilibrated in 0.1% (v/v) Optima TFA (Fisher) in LC-MS Optima water (Fisher). The separation consisted of washing with 100% A (0.1% [v/v] TFA in Optima water) from 0 to 3 min, followed by a linear gradient from 0 to 20% B (0.1% [v/v] TFA in Optima acetonitrile) from 3 to 20 min, followed by a linear gradient from 20% B to 100% B from 20 to 23 min, washing with 100% B from 23 to 26.5 min, and re-equilibration into 100% A from 26.5 to 30.1 min.