Project description:<p>We generated a collection of patient-derived pancreatic normal and cancer organoids. We performed whole genome sequencing, targeted exome sequencing, and RNA sequencing on organoids as well as matched tumor and normal tissue if available. This dataset is a valuable resource for pancreas cancer researchers, and those looking to compare primary tissue to organoid culture. In our linked publication, we show that pancreatic cancer organoids recapitulate the mutational spectrum of pancreatic cancer. Furthermore, RNA sequencing of organoids demonstrates the presence of both transcriptional subtypes of pancreas cancer.</p>
Project description:Methionine plays a critical role in various biological and cell regulatory processes, making its chemoproteomic profiling indispensable for exploring its functions and potential in protein therapeutics. However, the advancement of methionine chemoproteomics is currently hindered by the scarcity of efficient labeling strategies. Methionine exhibits high kinetic reactivity and undergoes rapid oxidation due to the lower energy gap between Lowest Unoccupied Molecular Orbital (LUMO) and Highest Occupied Molecular Orbital (HOMO). Building on this unique high reactivity of methionine (Met), we introduce Copper(I)-Nitrene Platform (CuNiP) for robust, and selective labeling of Met to generate highly stable sulfonyl sulfimide conjugates under physiological conditions. We demonstrated the versatile proficiency of CuNiP to label methionine in a wide range of bioactive peptides, intact proteins (6.5-79.5 kDa) independent of molecular weight and 3D structures and proteins in complex cell lysate mixtures with varying payloads. Leveraging nitrene reactivity-based probes, we have discovered an array of new, previously undiscovered, ligandable proteins and sites harboring hyperreactive methionine within the landscape of the human proteome. Crucially, we employed CuNiP for live cell chemoproteomic profiling of methionine in breast (T47D) and prostate cancer (LnCap) cell lines, observing minimal cytotoxic effects and achieving dose-dependent labeling. Confocal imaging further revealed the spatial distribution of modified proteins within cell membrane, cytoplasm, and nucleus, underscoring the platform's potential in profiling the cellular interactome. This breakthrough, with its insights into live cell methionine labeling will deepen our understanding of protein biochemistry and also pioneers a transformative pathway in drug discovery.
Project description:DNA-binding proteins are promising therapeutic targets but notoriously difficult to drug. Here, we evaluate a chemoproteomic DNA interaction platform as a complementary strategy for parallelized compound profiling. To enable this approach, we determined the proteomic binding landscape of 92 immobilized DNA sequences. Perturbation-induced activity changes of captured transcription factors in disease-relevant settings demonstrated functional relevance of the enriched sub-proteome. Chemoproteomic profiling of >300 cysteine-directed compounds against a coverage optimized bead mixture, which specifically captures >150 DNA binders, revealed competition of several DNA-binding proteins, including the transcription factors ELF1 and ELF2. We also discovered the first compound which displaces the DNA-repair complex MSH2-MSH3 from DNA. Compound binding to cysteine 252 on MSH3 was confirmed using chemoproteomic reactive cysteine profiling. Overall, these results suggested that chemoproteomic DNA bead pull-downs enable the specific read-out of transcription factor activity and can identify functional “hot-spots” on DNA binders towards expanding the druggable proteome.
Project description:Current methods for measuring amino-acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts, and software to boost data acquisition in real-time on the mass spectrometer. Our method, Streamlined Cysteine Activity-Based Protein Profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites in 18 min per compound. We applied it to identifying the proteome-wide targets of a covalent inhibitor of mutant KRASG12C and of ibrutinib, a covalent inhibitor of BTK. In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines , which includes >20,000 cysteines in >6,000 proteins per cell line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.
Project description:Chemoproteomics investigates small molecule-protein interactions and has made significant progress in recent years. Despite its vast potential, the proteome-wide profiling of reactive cysteine ligandability remains a formidable task to adapt for high throughput applications. This is primarily due to a lack of platforms capable of achieving the desired depth using low sample input in 96- or 384-well plates. Here we have revamped the cysteine profiling platform to address the challenge with an eye toward performing high-throughput library screening in plates. By incorporating several changes including i) an 18-plex TMT sample multiplexing strategy, ii) a magnetic beads-based one-pot workflow, iii) a 10X higher capacity streptavidin resin, and iv) optimized mass spectrometry analyses, a plate-based platform was developed that enables routine interrogation of either ~18,000 or ~24,000 reactive cysteines based on starting amounts of 10 or 20 µg, respectively. We applied the platform to screen a library of 192 electrophiles in the native HEK293T proteome, mapping the ligandablity of 38,450 reactive cysteines from 8,274 human proteins. The significantly improved depth revealed many previously unknown reactive cysteines and cysteine-ligand interactions and led to the identification of an azepane-containing acrylamide which has preferential binding to cysteines in EGF-like domains. We further applied the platform to characterize new cellular targets of well-studied compounds and covalent drugs in three different human cell lines. We found that ARS-1620, a KRASG12C inhibitor, also binds to cysteine 140 of an off-target adenosine kinase ADK, inhibiting its kinase activity. The platform represents a major step forward to high throughput evaluation of reactive cysteines on a proteome-wide scale.
Project description:Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive frag-ments, drugs and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole as a cyste-ine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cyste-ine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage thus enabling profiling of cysteines directly in live cells. Further, the fluorine ‘signature’ of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (−)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analog target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for non-homologous end joining (NHEJ) upon DNA double strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light induced DNA damage. Altogether, our next generation cysteine-reactive probe enables broader and deeper profiling of the cyste-inome rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.
Project description:Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5,500 cysteine sites across ~3,000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.
Project description:The major histocompatibility complex class I (MHC-I) antigen presentation pathways play pivotal roles in orchestrating immune responses. Recent studies have begun to utilize cysteines within the immunopeptidome for therapeutic applications, such as using covalent ligands to create haptenated neoantigens for inducing an immune response. In this study, we report a platform for mapping reactive cysteines present on MHC-I-bound peptide antigens. We have developed cell-impermeable sulfonated maleimide probes capable of effectively capturing reactive cysteines on antigens. Utilizing these probes in chemoproteomic experiments, we discovered that cysteines on MHC-I-bound antigens exhibit various degrees of reactivity. Furthermore, interferon-gamma stimulation produces increased reactivity of cysteines at position 8 of 9-mer MHC-I-bound antigens. Our findings may open up new avenues for understanding the distinctive roles of cysteine within the MHC-I immunopeptidome and leveraging the differentially reactive cysteines for therapeutic intervention.