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Ligand observed MS screening and validation of the fragment libraryA two stage fragment screening strategy based on the ligand observed MS analysis was devised that involved a primary screen of 384 fragment as a whole followed by a secondary independent screen of fragment cocktails of less complexity. In the primary screen, a mixture of all 384 fragments was incubated with NS5B protein before the fragment bound complexes were separated from excessive free fragments using ultrafiltration. Notably, purification of protein complexes in solution by ultrafiltration eliminates the need of protein immobilization and thus preserves the native conformation of the target protein during its interaction with fragments. Fragments dissociated from the target using 90% methanol in deionized water were subjected to LC MS analysis and those showing MS intensity substantially higher than the negative control (protein free incubation) were considered positive hits (Figure 1A). Although in our previous assay development we justified an S/N ratio of 2 to be the threshold for positive binding25, here we raised the S/N threshold to 10 so as to retain the upmost strong binders given that low affinity fragments are more inclined to nonspecific adsorption. Impressively, the high selectivity of high resolution MS (HRMS) analysis enabled rapid screening of the 384 fragment mixture within 20 min analytical time, generating 20 preliminary hits with S/N above 10 (Figure 1B, full data sets in Table S1). It is noted that 20 compounds (5%) in this library are not amenable to our analysis, probably due to the limited sensitivity of the mass spectrometer used in this study or the intrinsically low ionization efficiency of these compounds. Nevertheless, these undetectable fragments were still included in the screening cocktail to maximize the complexity of multiple ligand interactions during incubation. To the best of our knowledge, such a high capacity in mixture screening has never been shown before for fragment libraries, and we anticipate the throughput can be easily boosted to screening fragments within half an hour using a state of the art mass spectrometer.                                                                                                                                                                                                                                                                                                                                

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