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

Abstract

Fast and efficient handling of ligands and biological targets are required in bioaffinity screening based on native electrospray ionization mass spectrometry (ESI-MS). We use a prototype microfluidic autosampler, called the "gap sampler", to sequentially mix and electrospray individual small molecule ligands together with a target protein and compare the screening results with data from thermal shift assay and surface plasmon resonance. In a first round, all three techniques were used for a screening of 110 ligands against bovine carbonic anhydrase II, which resulted in five mutual hits and some false positives with ESI-MS presumably due to the high ligand concentration or interferences from dimethyl sulfoxide. In a second round, 33 compounds were screened in lower concentrations and in a less complex matrix, resulting in only true positives with ESI-MS. Within a cycle time of 30 s, dissociation constants were determined within an order of magnitude accuracy consuming only 5 pmol of ligand and less than 15 pmol of protein per screened compound. In a third round, dissociation constants of five compounds were accurately determined in a titration experiment. Thus, the gap sampler can rapidly and efficiently be used for high-throughput screening.

Analysis of histidine‐tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry

“…Methods for high‐throughput protein expression and functional protein analysis have increasingly become popular in the post‐genomic era, leading to the design of multiple direct MS‐based platforms capable of analysis of label‐free enzymatic activity, 43–45 inhibition 46,47 and ligand binding 48–50 using purified enzymes. Another commonly used method for investigating enzymatic activity is protein microarray 51,52 .…”

Determination of dissociation constants via quantitative mass spectrometry

“…This can be done either by preparation of a titration series or with a continuous ESI source, which allows the gradual combination of the two components (Fryčák and Schug, 2007;Bui et al, 2022). The latter is for example used in the "gap sampler" by the Zenobi group for bioaffinity screening with high-throughput and low sample consumption (Neu et al, 2013;Kaeslin et al, 2021). Additionally, the comparison of peak intensities of the complexes vs. the unbound molecule as a function of the varied concentration allows a determination of the thermodynamic equilibrium as well as the ratio of the response factors of the bound and the unbound species (Gabelica et al, 2003).…”