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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Microfluidics

Background:

• Bioaffinity screening requires efficient methods for handling ligands and biological targets.
• Native electrospray ionization mass spectrometry (ESI-MS) is a key technique for such analyses.
• Existing methods can be limited by sample consumption and processing time.

Purpose of the Study:

• To introduce and evaluate a prototype microfluidic autosampler, the "gap sampler," for bioaffinity screening.
• To compare the performance of the gap sampler with established techniques like thermal shift assay and surface plasmon resonance.
• To assess the efficiency, accuracy, and throughput of the gap sampler for determining ligand-protein interactions.

Main Methods:

• Utilized a prototype microfluidic autosampler ("gap sampler") for sequential mixing and electrospraying of ligands and target proteins.
• Performed bioaffinity screening using native electrospray ionization mass spectrometry (ESI-MS).
• Validated results by comparing ESI-MS data with thermal shift assay and surface plasmon resonance.

Main Results:

• Initial screening of 110 ligands against bovine carbonic anhydrase II identified five mutual hits, with some false positives in ESI-MS at high ligand concentrations.
• Second-round screening of 33 compounds at lower concentrations and in a simpler matrix yielded only true positives with ESI-MS.
• Dissociation constants were determined with order-of-magnitude accuracy within 30 seconds per cycle, using only 5 pmol of ligand and 15 pmol of protein per compound.
• Accurate dissociation constants were confirmed for five compounds via titration experiments.

Conclusions:

• The gap sampler enables rapid and efficient bioaffinity screening using native ESI-MS.
• Optimized conditions (lower concentrations, simpler matrix) improve ESI-MS accuracy in ligand-protein interaction studies.
• The gap sampler significantly reduces sample consumption and analysis time, facilitating high-throughput screening.