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

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.

Automated and enhanced extraction of a small molecule-drug conjugate using an enzyme-inhibitor interaction based SPME tool followed by direct analysis by ESI-MS.

We report a novel, fast, and automatic SPME-based method capable of extracting a small molecule-drug conjugate (SMDC) from biological matrices. Our method relies on the extraction of the drug conjugate followed by direct elution into an electrospray mass spectrometer (ESI-MS) source for qualitative and quantitative analysis. We designed a tool for extracting the targeting head of a recently synthesized SMDC, which includes acetazolamide (AAZ) as high-affinity ligand specific to carbonic anhydrase IX. Specificity of the extraction was achieved through systematic optimization. The design of the extraction tool is based on noncovalent and reversible interaction between AAZ and CAII that is immobilized on the SPME extraction phase. Using this approach, we showed a 330% rise in extracted AAZ signal intensity compared to a control, which was performed in the absence of CAII. A linear dynamic range from 1.2 to 25 µg/ml was found. The limits of detection (LOD) of extracted AAZ from phosphate-buffered saline (PBS) and human plasma were 0.4 and 1.2 µg/ml, respectively. This with a relative standard deviation of less than 14% (n = 40) covers the therapeutic range. Graphical abstract.

The capillary gap sampler, a new microfluidic platform for direct coupling of automated solid-phase microextraction with ESI-MS.

A new technology for rapid, automated coupling of solid-phase microextraction and mass spectrometry is introduced. Use of a so-called capillary gap sampler for automated solid-phase microextraction and direct delivery of the extracted analytes to a mass spectrometer provides certain advantages over existing technologies: coupling of the capillary gap sampler to a mass spectrometer offers quick, automated, and site-specific extraction from very low volume samples. High stability, reusability, and repeatability were achieved through systematic optimization. Diazepam, oxazepam, and nordiazepam were used as test compounds in all experiments. The ability of the sampler to extract benzodiazepines from human plasma (limit of detection 0.3 µg/mL) in the therapeutic range was confirmed. A linear dynamic range from 1 to 1000 ng/mL for all three analytes was found. The relative standard deviation of 20 extractions was between 11% and 17%, for oxazepam, nordiazepam, and diazepam, indicating acceptable repeatability of the method. Graphical Abstract Schematic of the desorption step inside the liquid bridge formed between two capillaries in thecapillary gap sampler.