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

Abstract

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.

A Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

“…This approach provides advantages of reduced instrument time, efforts and cost by overcoming the need for library generation. 26 The data was also searched using library-free mode in DIA-NN which instead generates an in silico library from a protein sequence database (Figure 2, 4) . 36 While their performance have previously been benchmarked and shown unique advantages in large-scale proteomic and phosphoproteomic workflows, they have never been compared for TPP.…”

“…25 More recently, it was adopted in a matrix thermal shift assay to detect ligand concentration-dependent stabilisation of proteins, at a single melting temperature. 26 Ruan et al treated K562 lysates with staurosporine, a model widely used for thermal shift assay method evaluation and demonstrated reduction in sample preparation time, cost and effort. By adopting a library-free, DirectDIA approach, they increased throughput and achieved comparable sensitivity for identifying kinase targets to a recent 2D-TPP TMT study.…”

“…By adopting a library-free, DirectDIA approach, they increased throughput and achieved comparable sensitivity for identifying kinase targets to a recent 2D-TPP TMT study. 13,26 There are now various pipelines for DIA peptide identification, each with distinct strengths and suitability depending on the type of proteomic data. [27][28][29] Project-specific libraries generated by analysing pre-fractionated DDA samples, which are representative of the model system, historically provide the greatest proteome coverage but largely depend on the size and quality of the library, increasing instrument run-time.…”

“…By adopting a library-free, DirectDIA approach, they increased throughput and achieved comparable sensitivity for identifying kinase targets to a recent 2D-TPP TMT study. 13 , 26…”

Profiling protein targets of cellular toxicant exposure

“…159 A recent report found that data independent analysis (DIA) also provides good quantitative precision. 160 Although the melting temperature is a proxy for protein stability, it does not represent an equilibrium measurement of unfolding. Hence, unlike the critical chaotrope concentration in SPROX or other footprinting techniques, melting temperature does not allow direct determination of Δ G unfolding .…”