Dataset Information

Global profiling of AMG510 modified proteins identified tumor suppressor KEAP1 as an off-target

ABSTRACT: Here, we presented a streamlined workflow to measure abundant AMG510 modified peptides including that of KRAS/G12C by direct profiling, and a pan-AMG510 antibody peptide IP workflow to profile less abundant AMG510 off-targets.

ORGANISM(S): Homo Sapiens

SUBMITTER: Jun Qin

PROVIDER: PXD029449 | iProX | Wed Jan 11 00:00:00 GMT 2023

REPOSITORIES: iProX

ACCESS DATA

Publications

Global profiling of AMG510 modified proteins identified tumor suppressor KEAP1 as an off-target.

Wang Yini Y Zhong Bowen B Xu Caixia C Zhan Dongdong D Zhao Songhao S Wu Hongxing H Liu Mingwei M Lan Xiaoling X Cai Danni D Ding Qian Q Zheng Biao B Lan Jiong J Lv Qiang Q Wang Yi Y Qin Jun J

iScience 20230128 2

KRAS inhibitor AMG510 covalently modifies the G12C residue and inactivates the KRAS/G12C function. Because there are many reactive cysteines in the proteome, it is important to characterize AMG510 on-target modification and off-targets. Here, we presented a streamlined workflow to measure abundant AMG510 modified peptides including that of KRAS/G12C by direct profiling, and a pan-AMG510 antibody peptide IP workflow to profile less abundant AMG510 off-targets. We identified over 300 off-target si ...[more]

PMID: 36824285

Similar Datasets

Project description:The precise regulation of transcription requires the coordinated activity of numerous proteins and protein complexes. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unresolved. Accurately defining the direct effects of transcriptional regulators is critical to addressing these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly synthesized RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly synthesized RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we assembled an end-to-end workflow to deplete proteins of interest using the AID system and measure newly synthesized RNA using SLAM-seq in the model eukaryote, Saccharomyces cerevisiae. We provide an open-source, step-by-step protocol to support rapid implementation of this workflow. We include methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, mRNA-seq library construction, and data analysis. Additionally, we demonstrate that this workflow can help define the direct effects of transcriptional regulators using the bromodomain and extra-terminal domain (BET) proteins, Bdf1 and Bdf2, as an example. We provide evidence that data generated using this workflow is robust to normalization using whole-cell spike-in or total read counts, correlates well with 4tU-seq data, and identifies extensive differential expression due to the depletion of Bdf1 and Bdf2. Taken together, this workflow will help address outstanding questions underlying the molecular basis of transcriptional regulation and other processes in S. cerevisiae and other eukaryotes.

Dataset Information

Global profiling of AMG510 modified proteins identified tumor suppressor KEAP1 as an off-target

Publications

Global profiling of AMG510 modified proteins identified tumor suppressor KEAP1 as an off-target.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure