The current predominant theapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target’s ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000–6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

Isobaric mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive multiplexed peptide/protein quantification technique in mass spectrometry. However, accurate quantification of complex proteomic samples is impaired by cofragmentation of peptides, leading to systematic underestimation of quantitative ratios. Label-free quantification strategies do not suffer from such an accuracy bias but cannot be multiplexed and are less precise. Here, we compared protein quantification results obtained with these methods for a chemoproteomic competition binding experiment and evaluated the utility of measures of spectrum purity in survey spectra for estimating the impact of cofragmentation on measured TMT-ratios. While applying stringent interference filters enables substantially more accurate TMT quantification, this came at the expense of 30%-60% fewer proteins quantified. We devised an algorithm that corrects experimental TMT ratios on the basis of determined peptide interference levels. The quantification accuracy achieved with this correction was comparable to that obtained with stringent spectrum filters but limited the loss in coverage to <10%. The generic applicability of the fold change correction algorithm was further demonstrated by spiking of chemoproteomics samples into excess amounts of E. coli tryptic digests.

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.

Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease and non-selective inhibitors are protective in rodent disease models. Due to their poor potency and selectivity, the neuroprotective mechanism of these tool compounds has remained elusive so far and it is still unknown whether selective LRRK2 inhibition can attenuate mutant LRRK2-dependent toxicity in human neurons. Here, we employ a chemoproteomics strategy to identify potent, selective and metabolically stable LRRK2 inhibitors. We demonstrate that CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. These precise chemical probes further validate this emerging therapeutic strategy. They will enable more detailed studies of LRRK2-dependent signaling and pathogenesis and accelerate drug discovery.