Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment‐Based Screening

Abstract

Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention in recent years. So far, only a few studies have discussed mass spectrometry as a screening technology for fragments. Herein, we report the application of native electrospray ionization mass spectrometry (MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screening conducted with a thermal shift assay (TSA) and represented different binding categories. Our data indicated that, beside specific complex formation, many fragments show extensive multiple binding and also charge-state shifts. Both of these factors complicate automated data analysis and decrease the attractiveness of native MS as a primary screening tool for fragments. A comparison of the hits identified by native MS and TSA showed good agreement for two of the proteins. Furthermore, we discuss general challenges, including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS to be a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening.

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry

“…The same problem with false positives was also reported in the NanoMate HTS studies mentioned above. 24,25 Nonspecific binding might be enhanced at high L concentration or with the presence of DMSO, which was reported to alter the binding strength in ESI. 41 To reduce the effect of nonspecific binding, the first round positive hits found by TSA, SPR, and the gap sampler were rescreened in a second round (assuming the precipitating A29 to be binding, ignoring insoluble A98).…”

“…Using TSA and native MS with the NanoMate, Goẗh et al screened four different proteins against 33, 16, 21, or 21 compounds, respectively. 24 Depending on the protein, the agreement between the methods was high (16 mutual hits) to very poor (0 mutual hits). Later, 361 ligands were screened against endothiapepsin by Schiebel et al with a biochemical assay, a reporter ligand displacement assay, NMR, TSA, MST, X-ray crystallography, and native MS with the NanoMate.…”

Label‐free Bio‐affinity Mass Spectrometry for Screening and Locating Bioactive Molecules

“…However, significant deviations in the results are also rather common, both between native MS and other methods as well as between different research laboratories. For instance, Göth et al (2017) applied native MS and TSA to perform fragment‐based screening against four target proteins, including MTH1, KDM5B, BRPF1, and UHRF1. Interestingly, the hit overlap rates for MTH1 and KDM5B are 100% and 80%, whereas this quite good agreement is not observed for BRPF1 and UHRF1.…”

Native mass spectrometry for the design and selection of protein bioreceptors for perfluorinated compounds

“…The Kd of untreated hSA-PFOA complex was estimated from a single point measurement, as described by Göth et al, for all charge states and the mean Kd was reported. 42 Arrival times were extracted at the full width at half maximum from the whole peak, including salt adducts and complexes (where appropriate). This corresponds to an m/z-window from 4435 to 4455 m/z and 4435 to 4520 m/z for the unbound and bound hSA, respectively.…”