Figure 1
Fragment library screening based on the ligand-observed LC/MS approach.
(A) Scheme of the MS-based fragment screening workflow. The protein is incubated with a mixture of the fragment library, ligand-bound complexes are purified by ultrafiltration and the dissociated ligands are identified and quantified by LC/MS. Positive fragment binding is indicated by its S/N ratio > 10. (B) Results of the primary screen of a 384-member fragment library in a single run. Solid and open circles designate data points acquired in the positive and negative modes of MS analysis respectively. Circles above the threshold line denote positive hits. S/N ratios are averages of experimental duplicates. (C) Results from the secondary screen of 7 subdivided fragment cocktails, each consisting of 50 or 86 members. A total of 12 hits were identified in this run. S/N ratios are averages of experimental duplicates. (D) Binding degrees of individual fragments measured from the 12-hit mixture at different protein/ligand ratios (P/L) while the protein concentration was held constant in incubation. Ten fragments were validated to bind to NS5B and their binding degrees indicate a relative order of binding affinity. Error bars denote s.d. from experimental duplicates.
