Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis

Abstract

Summary Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed “multiplexed proteome dynamics profiling” (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.

Isoform‐resolved correlation analysis between mRNA abundance regulation and protein level degradation

“…We have compared pSILAC‐DIA to two alternative workflows, pSILAC‐MS1 (Schwanhausser et al , ; Mathieson et al , ) and pSILAC‐TMT (e.g., when combined with synchronous precursor selection (SPS)‐based MS3 scanning for improving the TMT quantification, see Appendix; McAlister et al , ; Welle et al , ; Savitski et al , ; Zecha et al , ). We identified particular advantages of pSILAC‐DIA over the existing methods, such as lower charge states of MS2 signals which reduced MS data processing difficulty (Appendix Fig S2), significantly better accuracy in quantifying SILAC heavy‐to‐light (H/L) ratios than pSILAC‐MS1 (whose performance was shown to be comparable to pSILAC‐TMT (Zecha et al , ); Appendix Figs S3 and S4), much more quantitative data points with the potential to use most high‐resolution heavy and light fragment ions (Appendix Fig S5) together with their elution traces along the liquid chromatography (Appendix Fig S6), as well as other flexibilities and potentials.…”

Thermal proteome profiling for interrogating protein interactions

“…More commonly, TPP experiments involve chemical [e.g., drug (Azimi et al, 2018;Becher et al, 2016;Hu et al, 2019;Huber et al, 2015;Kitagawa et al, 2017;Mateus et al, 2018Mateus et al, , 2016Reinhard et al, 2015;Savitski et al, 2014Savitski et al, , 2018 . Some of the perturbations can be applied in a dose-dependent manner (Becher et al, 2016) or time-dependent manner Dai et al, 2018) to improve data analysis or facilitate mechanistic understanding of the perturbation (Fig 2).…”

The PROTAC technology in drug development

“…68 Further studies showed that PROTAC ARV-771 treatment reduced leukaemia burden and improved survival of HEL92.1.7 cellsengrafted NSG mice, better than the effect from OTX015. 75 A recent study extended designs of PROTACs against TRIM24, another bromodomain-containing transcriptional regulator. Notably, they showed that PROTACs overcame the drug resistance from bortezomib, dexamethasone, lenalidomide, and pomalidomide.…”