A Dimethyl-Labeling-Based Strategy for Site-Specifically Quantitative Chemical Proteomics

Yang, Fan; Gao, Jinjun; Che, Jinteng; Jia, Guogeng; Wang, Chu

doi:10.1021/acs.analchem.8b02426

Cited by 60 publications

(60 citation statements)

References 32 publications

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“…The quantitative chemical proteomics was performed as recently described 42 peptides were also restricted to fully tryptic with a defined peptide false positive rate of 1%. The ratios (L/H) of reductive dimethylation were quantified by the CIMAGE software as described before 45 .…”

Section: Cys Reactivity Validation Through Iodoacetyl Modified Probe mentioning

confidence: 99%

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Pan

Zheng

et al. 2020

Preprint

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p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis 1 . Human p97 mutations are associated with several neurodegenerative diseases 2,3 . Targeting p97 and its cofactors is a strategy for cancer drug development 4 . Despite significant structural insights into the fungal homolog Cdc48 5-7 , little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97 8 , but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis.The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that under oxidative conditions, the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) inhibits p97 function by releasing cupric ions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.Recently, the cryo-electron microscopy (cryo-EM) structures of the thermophilic fungus Cdc48-Npl4/Ufd1 complex 6 and the yeast Cdc48-Npl4/Ufd1-Eos complex 5 were reported. In

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Section: Cys Reactivity Validation Through Iodoacetyl Modified Probe mentioning

confidence: 99%

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Pan

Zheng

et al. 2020

Preprint

Self Cite

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“…Previous reports indicated that the application of multiplexed labeling strategies for cleavable linkers could result in the identification of several thousand unique peptides within a single experiment when using reductive dimethyl and iTRAQ labeling strategies 47,48 . We therefore decided to implement a recently published protocol 49 utilizing TMT labeling in combination with the DADPS linker (Supp Fig 3A).…”

Section: Resultsmentioning

confidence: 99%

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

Rabalski

Bogdan

Baranczak

2019

Preprint

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Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely-used chemically-cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, a mass tolerant database search strategy using MSFragger software was performed. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.

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“…To overcome the deficiencies in-gel separation and band identification, quantitative methods have been incorporated into chemical proteomics. 40,[115][116][117][118] By comparing the relative abundance of proteins between different samples or against appropriate negative controls, proteins with a higher abundance ratio (>1.5 or 2, different values have been used) than a threshold are identified as specific targets, thus avoiding nonspecific binders.…”

Section: Quantitative Proteomicsmentioning

confidence: 99%

Target identification of natural medicine with chemical proteomics approach: probe synthesis, target fishing and protein identification

Chen

Wang

et al. 2020

Sig Transduct Target Ther

140

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Natural products are an important source of new drugs for the treatment of various diseases. However, developing natural productbased new medicines through random moiety modification is a lengthy and costly process, due in part to the difficulties associated with comprehensively understanding the mechanism of action and the side effects. Identifying the protein targets of natural products is an effective strategy, but most medicines interact with multiple protein targets, which complicate this process. In recent years, an increasing number of researchers have begun to screen the target proteins of natural products with chemical proteomics approaches, which can provide a more comprehensive array of the protein targets of active small molecules in an unbiased manner. Typically, chemical proteomics experiments for target identification consist of two key steps: (1) chemical probe design and synthesis and (2) target fishing and identification. In recent decades, five different types of chemical proteomic probes and their respective target fishing methods have been developed to screen targets of molecules with different structures, and a variety of protein identification approaches have been invented. Presently, we will classify these chemical proteomics approaches, the application scopes and characteristics of the different types of chemical probes, the different protein identification methods, and the advantages and disadvantages of these strategies.

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A Dimethyl-Labeling-Based Strategy for Site-Specifically Quantitative Chemical Proteomics

Cited by 60 publications

References 32 publications

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

Target identification of natural medicine with chemical proteomics approach: probe synthesis, target fishing and protein identification

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