Implementing Thermal Proteome Profiling as a tool to identify off-targets of AMPK modulators using LC-MS/MS
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- Thermal Proteome Profiling (TPP) is a liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based workflow for performing proteome-wide analyses of protein-ligand interactions. This is achieved through the monitoring of protein thermal stability changes across a temperature range (TPP-TR) upon ligand binding. Among the different types of protein-ligand interactions that can be interrogated with TPP, a common field of interest is the identification of direct targets of protein-targeting drugs, including both unknown beneficial targets and unintended, potentially harmful off-targets. This research project aimed to implement a standard TPP workflow in the host laboratory and use it to identify potential (off-)targets of modulators of the AMP-activated protein kinase (AMPK), namely the activator MK-8722 and the inhibitor BAY-3827. A tailored TPP workflow was first established, including TPP-TR, LC-MS/MS using tandem mass tags (TMT), and bioinformatic analysis. Our TPP-TR sample preparation protocol was validated by immunoblotting and LC-MS/MS on lysates of wild-type mouse embryonic fibroblasts (WT MEF) using Trichostatin A, a histone deacetylase (HDAC) inhibitor known to cause thermal stabilisation of HDAC1. Relative quantification by LC-MS/MS was achieved using TMT labelling. Validation by immunoblotting was performed in parallel on lysates of freshly isolated mouse hepatocyte as a more physiologically relevant model. Our validated methods were then applied on WT MEF lysates treated with AMPK modulators MK-8722 and BAY-3827. Immunoblotting displayed potential compound-induced thermal shifting of AMPK subunits and enabled refining the compound concentrations. Results were expanded proteome-wide with LC-MS/MS to quantitatively evaluate AMPK thermal shifting and identify potential off-targets of MK-8722 and BAY-3827.The data processing pipeline was based on the open-source “TPP” R package for proteome-wide analysis of melting curves and melting temperature shifts. Potential off-targets identified in WT MEF lysates were confronted to those identified in MEF lysates lacking active AMPK as a negative control. The established TPP workflow will be applied to other protein-targeting drugs which are already in clinical use. This could contribute to refining their characterisation and, hopefully, elucidate the origin of some of their adverse effects.