Interaction profiling methods to map protein and pathway targets of bioactive ligands

Abstract

Recent advances in -omic profiling technologies have ushered in an era where we no longer want to merely measure the presence or absence of a biomolecule of interest, but instead hope to understand its function and interactions within larger signaling networks. Here, we review several emerging proteomic technologies capable of detecting protein interaction networks in live cells and their integration to draft holistic maps of proteins that respond to diverse stimuli, including bioactive small molecules. Moreover, we provide a conceptual framework to combine so-called 'top-down' and 'bottom-up' interaction profiling methods and ensuing proteomic profiles to directly identify binding targets of small molecule ligands, as well as for unbiased discovery of proteins and pathways that may be directly bound or influenced by those first responders. The integrated, interaction-based profiling methods discussed here have the potential to provide a unique and dynamic view into cellular signaling networks for both basic and translational biological studies.

Keywords: Chemical probes; Proteomics; Systems biology; Target identification.

Figure 1.. Top-Down Protein Interaction Methods.

Mechanistic overview of “Top-Down”, global protein interaction profiling methods. A) General workflow of MS-based thermal profiling (e.g., TPP) to measure aggregate proteoform stability in live cells (i.e. the bulk population of a protein of interest). Bioactive ligands can cause altered protein stability, either due to direct ligand engagement or downstream effects on interactions with other biomolecules. B) Hotspot Thermal Profiling (HTP) combines posttranslational modification-specific enrichment with peptide-level thermal profiling to measure the stability of distinct modiforms, in contrast to TPP, which measures aggregate stability from many peptides in a protein of interest. HTP therefore permits interrogation of ligand-induced stability changes in native protein sub-populations directly in cells. C) Schematic depiction limited-proteolysis profiling (e.g., DARTS or LiP-MS). Ligand binding in lysates leads to stabilization or protection of specific protein surfaces against proteolysis. Quantitative LC-MS/MS detection and peptide-level mapping can identify proteins and specific sites that are protected, and thus candidate ligand binding sites.

Figure 2.. Bottom-Up, Proximity Profiling Methods.

A) Schematic of proximity labeling-based LC-MS/MS approaches to study protein-protein interactions, complexes, and protein localization. Labeling strategies can be classified in two distinct categories based on whether the technique produces a diffusible probe with a reactive group or whether it involves direct ligation of the tag onto the interacting proteins. B) Overview of intracellular proximity methods that rely on production of a diffusible reactive probe, with key assay parameters listed.

Figure 3:. Kinetic interaction profiling to identify small molecule target proteins and signaling networks.

Schematic depiction of cellular protein networks responding to a perturbation, in this case a bioactive small molecule. The direct (red) and secondary (blue) targets of the stimulus would theoretically be the most significantly altered proteins in top-down, and subsequent bottom-up profiling datasets in early timepoints, as depicted in hypothetical volcano plots. Subsequent timepoints can capture signal propagation as interactions ripple outwards through affected protein networks and pathways. Integrated tracking of proteome-wide interactions and phenotypic consequences could provide complementary and novel insights into small molecule mechanism(s) of action as well as basic topology of interconnected proteins and pathways.