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Current protocols in chemical biology最新文献

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Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes 同位素靶向糖蛋白组学(IsoTaG)表征复杂蛋白质组中完整的代谢标记糖肽
Current protocols in chemical biology Pub Date : 2016-03-16 DOI: 10.1002/9780470559277.ch150185
Christina M. Woo, Carolyn R. Bertozzi
{"title":"Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes","authors":"Christina M. Woo,&nbsp;Carolyn R. Bertozzi","doi":"10.1002/9780470559277.ch150185","DOIUrl":"10.1002/9780470559277.ch150185","url":null,"abstract":"<p>Protein glycosylation plays many critical roles in biological function and creates the most diversity of all post-translational modifications (PTMs). Glycan structural diversity is directly correlated with difficulty in characterizing the intact glycoproteome by mass spectrometry (MS). In this protocol, we describe a novel mass-independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG). To use IsoTaG, cell culture samples are metabolically labeled with an azido- or alkynyl-sugar. Metabolically labeled glycoproteins are then tagged using Click chemistry and enriched with an isotopic recoding biotin probe. Intact glycopeptides are recovered by cleavage of the probe, analyzed with directed MS, and assigned by targeted mass-independent data analysis. The outlined procedure is well defined in cell culture and has been executed with over 15 cell lines. © 2016 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50659567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 15
RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells 二聚体西兰花在活细菌和哺乳动物细胞中的RNA成像
Current protocols in chemical biology Pub Date : 2016-03-16 DOI: 10.1002/9780470559277.ch150174
Grigory S. Filonov, Samie R. Jaffrey
{"title":"RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells","authors":"Grigory S. Filonov,&nbsp;Samie R. Jaffrey","doi":"10.1002/9780470559277.ch150174","DOIUrl":"10.1002/9780470559277.ch150174","url":null,"abstract":"<p>RNA spatial dynamics play a crucial role in cell physiology, and thus the ability to monitor RNA localization in live cells can provide insight into important biological problems. This unit focuses on imaging RNAs using an RNA mimic of GFP. This approach relies on an RNA aptamer called dimeric Broccoli, which binds to and switches on the fluorescence of DFHBI, a small molecule mimicking the fluorophore in GFP. Dimeric Broccoli is tagged to heterologously expressed RNAs and, upon DFHBI binding, the fluorescent signal of dimeric Broccoli reports the transcript's localization in cells. This protocol describes the process of validating the fluorescence of dimeric Broccoli−labeled transcripts in vitro and in cells, flow cytometry analysis to determine overall fluorescence levels in cells, and fluorescence imaging in bacterial and mammalian cells. Overall, the protocol should be useful for researchers seeking to image high-abundance RNAs, such as those transcribed off the T7 promoter in bacteria or off Pol III−dependent promoters in mammalian cells. © 2016 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470559277.ch150174","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50658900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 41
Chemical Synthesis of Ubiquitin Chains 泛素链的化学合成
Current protocols in chemical biology Pub Date : 2015-12-02 DOI: 10.1002/9780470559277.ch150099
Hosahalli P. Hemantha, Somasekhar Bondalapati, Sumeet K. Singh, Ashraf Brik
{"title":"Chemical Synthesis of Ubiquitin Chains","authors":"Hosahalli P. Hemantha,&nbsp;Somasekhar Bondalapati,&nbsp;Sumeet K. Singh,&nbsp;Ashraf Brik","doi":"10.1002/9780470559277.ch150099","DOIUrl":"10.1002/9780470559277.ch150099","url":null,"abstract":"<p>Chemical synthesis of complex biomolecules such as proteins is a challenging adventure, yet rewarding in driving various biochemical and biophysical research activities. Over the years, the refinement of peptide synthesis and invention of ligation methodologies have led to the successful synthesis of several complex protein targets. Ubiquitin bioconjugates, which are being studied intensively by many groups due to their involvement in numerous biological processes, represent a fine example where chemistry is greatly aiding these studies. In this article, we describe the synthetic routes and strategies to prepare different ubiquitin analogs with desired modifications, as well as di-ubiquitin chains. © 2015 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470559277.ch150099","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50658972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
A Bead-Based Proximity Assay for BRD4 Ligand Discovery 基于微球的BRD4配体发现接近试验
Current protocols in chemical biology Pub Date : 2015-12-02 DOI: 10.1002/9780470559277.ch150024
Justin M. Roberts, James E. Bradner
{"title":"A Bead-Based Proximity Assay for BRD4 Ligand Discovery","authors":"Justin M. Roberts,&nbsp;James E. Bradner","doi":"10.1002/9780470559277.ch150024","DOIUrl":"10.1002/9780470559277.ch150024","url":null,"abstract":"<p>Bromodomain-containing proteins have emerged as desirable targets for anti-neoplastic and anti-inflammatory drug discovery. Toward the development of selective inhibitors of the BET family of bromodomains, we optimized bead-based assays to detect interactions between bromodomains and poly-acetylated histone peptides. Donor and acceptor beads bound to target and ligand are brought into proximity by this protein-protein interaction. After laser illumination, singlet oxygen evolved from donor beads travels to the spatially close acceptor beads, resulting in chemiluminesence. This AlphaScreen assay has proven amendable to high-throughput screening, secondary validation, and specificity profiling during lead discovery and optimization. Here we report our protocol for assay development to measure inhibition of ligand binding to bromodomain-containing protein 4 (BRD4). We discuss the discovery of an appropriate probe, optimization of bead, probe, and protein concentrations, and the derivation of protein-probe inhibition curves. Finally, we explore the implementation of this technology for high-throughput screening of potential BRD4 inhibitors. © 2015 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470559277.ch150024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50659133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 18
Corking Nitrogen-Doped Carbon Nanotube Cups with Gold Nanoparticles for Biodegradable Drug Delivery Applications 用金纳米颗粒填充氮掺杂碳纳米管杯，用于可生物降解的药物输送应用
Current protocols in chemical biology Pub Date : 2015-12-02 DOI: 10.1002/9780470559277.ch150093
Seth C. Burkert, Alexander Star
{"title":"Corking Nitrogen-Doped Carbon Nanotube Cups with Gold Nanoparticles for Biodegradable Drug Delivery Applications","authors":"Seth C. Burkert,&nbsp;Alexander Star","doi":"10.1002/9780470559277.ch150093","DOIUrl":"10.1002/9780470559277.ch150093","url":null,"abstract":"<p>Carbon nanomaterials have been proposed as effective drug delivery devices; however their perceived biopersistence and toxicological profile may hinder their applications in medical therapeutics. Nitrogen doping of carbon nanotubes results in a unique “stacked-cup” structure, with cups held together through van der Waals forces. Disrupting these weak interactions yields individual and short-stacked nanocups that can subsequently be corked with gold nanoparticles, resulting in sealed containers for delivery of cargo. Peroxidase-catalyzed reactions can effectively uncork these containers, followed by complete degradation of the graphitic capsule, resulting in effective release of therapeutic cargo while minimizing harmful side effects. The protocols reported herein describe the synthesis of stacked nitrogen-doped carbon nanotube cups followed by effective separation into individual cups and gold nanoparticle cork formation resulting in loaded and sealed containers. © 2015 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50658808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
Molecular Evolution Directs Protein Translation Using Unnatural Amino Acids 分子进化指导蛋白质翻译使用非天然氨基酸
Current protocols in chemical biology Pub Date : 2015-12-02 DOI: 10.1002/9780470559277.ch150115
Vanessa E. Cox, Eric A. Gaucher
{"title":"Molecular Evolution Directs Protein Translation Using Unnatural Amino Acids","authors":"Vanessa E. Cox,&nbsp;Eric A. Gaucher","doi":"10.1002/9780470559277.ch150115","DOIUrl":"10.1002/9780470559277.ch150115","url":null,"abstract":"<p>Unnatural amino acids have in recent years established their importance in a wide range of fields, from pharmaceuticals to polymer science. Unnatural amino acids can increase the number of chemical groups within proteins and thus expand or enhance biological function. Our ability to utilize these important building blocks, however, has been limited by the inherent difficulty in incorporating these molecules into proteins. To address this challenge, researchers have examined how the canonical twenty amino acids are incorporated, regulated, and modified in nature. This review focuses on achievements and techniques used to engineer the ribosomal protein-translation machinery, including the introduction of orthogonal translation components, how directed evolution enhances the incorporation of unnatural amino acids, and the potential utility of ancient biomolecules for this process. © 2015 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50659171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Use of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways 利用人诱导多能干细胞衍生心肌细胞(hiPSC-CMs)监测心肌细胞信号通路的复合效应
Current protocols in chemical biology Pub Date : 2015-09-01 DOI: 10.1002/9780470559277.ch150035
Liang Guo, Sandy Eldridge, Mike Furniss, Jodie Mussio, Myrtle Davis
{"title":"Use of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways","authors":"Liang Guo,&nbsp;Sandy Eldridge,&nbsp;Mike Furniss,&nbsp;Jodie Mussio,&nbsp;Myrtle Davis","doi":"10.1002/9780470559277.ch150035","DOIUrl":"10.1002/9780470559277.ch150035","url":null,"abstract":"<p>There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. © 2015 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470559277.ch150035","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34141165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 25
Genetic Code Expansion of Mammalian Cells with Unnatural Amino Acids 利用非天然氨基酸扩展哺乳动物细胞的遗传密码
Current protocols in chemical biology Pub Date : 2015-09-01 DOI: 10.1002/9780470559277.ch150038
Kalyn A. Brown, Alexander Deiters
{"title":"Genetic Code Expansion of Mammalian Cells with Unnatural Amino Acids","authors":"Kalyn A. Brown,&nbsp;Alexander Deiters","doi":"10.1002/9780470559277.ch150038","DOIUrl":"10.1002/9780470559277.ch150038","url":null,"abstract":"The expansion of the genetic code of mammalian cells enables the incorporation of unnatural amino acids into proteins. This is achieved by adding components to the protein biosynthetic machinery, specifically an engineered aminoacyl‐tRNA synthetase/tRNA pair. The unnatural amino acids are chemically synthesized and supplemented to the growth medium. Using this methodology, fundamental new chemistries can be added to the functional repertoire of the genetic code of mammalian cells. This protocol outlines the steps necessary to incorporate a photocaged lysine into proteins and showcases its application in the optical triggering of protein translocation to the nucleus. © 2015 by John Wiley & Sons, Inc.","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34141164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Digestion, Purification, and Enrichment of Protein Samples for Mass Spectrometry 质谱分析中蛋白质样品的消化、纯化和富集
Current protocols in chemical biology Pub Date : 2015-09-01 DOI: 10.1002/9780470559277.ch140272
Victoria E. Hedrick, Mercedes N. LaLand, Ernesto S. Nakayasu, Lake N. Paul
{"title":"Digestion, Purification, and Enrichment of Protein Samples for Mass Spectrometry","authors":"Victoria E. Hedrick,&nbsp;Mercedes N. LaLand,&nbsp;Ernesto S. Nakayasu,&nbsp;Lake N. Paul","doi":"10.1002/9780470559277.ch140272","DOIUrl":"10.1002/9780470559277.ch140272","url":null,"abstract":"<p>Proteomic studies rely heavily on the use of liquid chromatography (LC)–mass spectrometry (MS and MS/MS) analyses to provide information about protein composition and function. Profiling the proteome can be the first step to understanding biological pathways, but the challenges scientists face with the complex nature of proteins and proteolysis products can be daunting. Techniques involving fractionation, immunoprecipitation, and phosphopeptide enrichment can simplify complex protein mixtures and enhance the amount of target proteins that are important to the investigator. Emphasis on sample preparation for LC-MS/MS analyses is essential to acquisition of high-quality data for proteomic research. Certain classes of reagents, materials, and contaminants that can be introduced during sample processing may limit the effectiveness of LC-MS/MS analysis. These protocols outline methods for proteolytic digestion of proteins that are compatible with LC-MS/MS, along with procedures that allow for simplification of complex protein matrices. © 2015 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470559277.ch140272","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34141166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 16
Biosensing with Virus Electrode Hybrids 利用病毒电极杂交体进行生物传感
Current protocols in chemical biology Pub Date : 2015-06-01 DOI: 10.1002/9780470559277.ch140213
Kritika Mohan, Reginald M. Penner, Gregory A. Weiss
{"title":"Biosensing with Virus Electrode Hybrids","authors":"Kritika Mohan,&nbsp;Reginald M. Penner,&nbsp;Gregory A. Weiss","doi":"10.1002/9780470559277.ch140213","DOIUrl":"10.1002/9780470559277.ch140213","url":null,"abstract":"<p>Virus electrodes address two major challenges associated with biosensing. First, the surface of the viruses can be readily tailored for specific, high affinity binding to targeted biomarkers. Second, the viruses are entrapped in a conducting polymer for electrical resistance-based, quantitative measurement of biomarker concentration. To further enhance device sensitivity, two different ligands can be attached to the virus surface, and increase the apparent affinity for the biomarker. In the example presented here, the two ligands bind to the analyte in a bidentate binding mode with a chelate-based avidity effect, and result in a 100 pM experimentally observed limit of detection for the cancer biomarker prostate-specific membrane antigen. The approach does not require enzymatic amplification, and allows reagent-free, real-time measurements. This article presents general protocols for the development of such biosensors with modified viruses for the enhanced detection of arbitrary target proteins. © 2015 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"7 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2015-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33982053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
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