Matrix Thermal Shift Assay for Fast Construction of Multidimensional Ligand–Target Space

Existing thermal shift-based mass
spectrometry approaches are able
to identify target proteins without chemical modification of the ligand,
but they are suffering from complicated workflows with limited throughput.
Herein, we present a new thermal shift-based method, termed matrix
thermal shift assay (mTSA), for fast deconvolution of ligand-binding
targets and binding affinities at the proteome level. In mTSA, a sample
matrix, treated horizontally with five different compound concentrations
and vertically with five technical replicates of each condition, was
denatured at a single temperature to induce protein precipitation,
and then, data-independent acquisition was employed for quick protein
quantification. Compared with previous thermal shift assays, the analysis
throughput of mTSA was significantly improved, but the costs as well
as efforts were reduced. More importantly, the matrix experiment design
allowed simultaneous computation of the statistical significance and
fitting of the dose–response profiles, which can be combined
to enable a more accurate identification of target proteins, as well
as reporting binding affinities between the ligand and individual
targets. Using a pan-specific kinase inhibitor, staurosporine, we
demonstrated a 36% improvement in screening sensitivity over the traditional
thermal proteome profiling (TPP) and a comparable sensitivity with
a latest two-dimensional TPP. Finally, mTSA was successfully applied
to delineate the target landscape of perfluorooctanesulfonic acid
(PFOS), a persistent organic pollutant that is hard to perform modification
on, and revealed several potential targets that might account for
the toxicities of PFOS.