Affinity Selection–Mass Spectrometry.

ALIS, the affinity selection–mass spectrometry platform that was used here to screen TrkA, is a dual-chromatography LC-MS system that incorporates a SEC column to separate mixtures of unbound compounds from protein-binding ligands, and then separates and identifies the bound ligands from the protein target using reversed-phase LC-MS. The ALIS system and methodologies used here have been previously described (28, 45, 46).

TrkA was screened against small molecules using ALIS as follows. TrkA protein was formulated at a concentration of 1.4 mg/mL (34 μM) in 50 mM Mes pH 6.5, 5 mM TCEP, 150 mM NaCl, 0.1% octyl-glucoside. The protein was separately diluted to an enzyme concentration of 10 μM with 59 mM Hepes pH 7.5 buffer containing 150 mM NaCl, 5.9 mM MgCl₂, and 0.25% n-octyl glucoside. For each ligand-binding reaction, 1.0 μL of 40× compound or compound mixtures was separately prediluted in 19 μL of 59 mM Hepes pH 7.5, 150 mM NaCl, 5.9 mM MgCl₂. One-microliter of diluted protein was then mixed with an equal volume of buffer-solubilized compound mixture in a 96-well plate. Final target concentration was set at 5 μM TrkA in 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT, 5 mM MgCl₂, 0.1% n-octyl glucoside, 2.5% (vol/vol) DMSO, and the final compound concentration was 1.0 μM per component for all reactions. After incubation at room temperature for 30 min, the samples, each containing a mixture of unbound compounds, protein–ligand complexes, and unliganded protein were cooled to 4 °C and then loaded onto an automated high-performance liquid chromatography system (Agilent 1100). Sample injections of 2 μL each were conducted on rapid SEC performed at 4 °C using a custom SEC column packed with proprietary gel-filtration media using a mobile phase of 0.7 M ammonium acetate pH 8 buffer running at a flow rate of 0.3 mL/min to rapidly separate protein–ligand complexes from unbound compounds. The eluent from the SEC column were directed through a UV detector (Agilent G1314A using a G1313 microflow cell) set at a UV absorbance of 230 nm to identify the protein–ligand complex. Upon detection, the complex was automatically diverted into a valve collection loop and then transferred to a reversed-phase chromatography column (Higgins Targa C18, 0.5 mm i.d × 50 mM length; Higgins Analytical Lab) running at 60 °C. Bound ligands are removed from the target protein, desalted, and eluted into the mass spectrometer using a solvent gradient of 0–95% (vol/vol) acetonitrile (0.1% formic acid) in water (0.1% formic acid) over 5 min using an Agilent G1376A capillary binary pump. Mass spectrometric detection was accomplished using a Waters LCT “Classic” or LCT “Premier” time-of-flight mass spectrometer operating in positive-ion ionization mode with a standard nebulized electrospray ionization source using a capillary voltage of 3.5 kV, desolvation temperature of 180 °C, source temperature of 100 °C, cone voltage set to 30 V, and extraction lens set to 3 V. Library mixtures that showed positive signals corresponding to calculated masses from known library components were repeated. Compounds of interest were each resynthesized as a single components, purified, and tested again against the TrkA target and also a negative control protein (Invertase) to rule out nonspecific binding. Those compounds that showed reproducible binding that were specific to the TrkA target were considered to be ALIS hits.