Cells are exposed to a test ligand or control vehicle, then heated to a precise temperature that causes unbound proteins to denature and aggregate while ligand-bound proteins remain soluble. The soluble fraction is recovered by centrifugation or solvent titration (with surfactants for membrane proteins), then denatured and proteolytically digested into peptides. Peptide mixtures undergo high-frequency full-scan nano-LC-MS, generating mass-to-charge, retention time, and intensity features. These features are uploaded to a cloud-based analysis platform, where isotope grouping, alignment across replicates, and statistical plus practical filters rank signals by differential intensity. Post-ranking MS/MS spectra are matched against protein databases to pinpoint proteins whose stability is altered by ligand binding.
Description
This technology stands out by quantifying all detectable MS signals before peptide identification, enabling an unbiased ranking of potential targets at a single temperature. Full-scan data acquisition at high frequency maximizes quantitative precision and dynamic range, while the use of surfactants and solvent titration broadens applicability to membrane and poorly soluble proteins. The cloud platform handles large datasets efficiently, offering scalable feature detection, statistical filtering, and database matching. Together, these aspects yield a rapid, sensitive approach for discovering both known and novel small-molecule–protein interactions.
Applications
- Drug target identification
- Small molecule off-target profiling
- Biomarker discovery and validation
- Membrane protein ligand screening
- Proteome-wide binding assays
Advantages
- Unbiased proteome-wide target identification using label-free full-scan mass spectrometry
- Single‐temperature thermal shift simplifies workflow and reduces assay time
- High sensitivity and dynamic range by prioritizing quantitative MS signals over MS/MS
- Compatible with membrane proteins via surfactant or solvent titration
- Quantitative ranking of ligand‐stabilized proteins enhances target prioritization
- Scalable, automated data processing and statistical filtering on the CHORUS cloud platform
- No labeling or protein modification required, preserving native protein context
IP Status
https://patents.google.com/patent/US11567074B2
