Current Protocols in Protein Science最新文献_第3页

Current Protocols in Protein Science Pub Date : 2019-09-12 DOI: 10.1002/cpps.81

{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpps.81","DOIUrl":"https://doi.org/10.1002/cpps.81","url":null,"abstract":"Cover: In Wolfe et al. (https://doi.org/10.1002/cpps.96), Representative two phases of the isothermal detergent desorption of membrane proteins. (A) The initial phase is detergent predesolvation. (B) The follow-up, time-dependent phase is detergent desolvation. Predesolvation results from the dissociation of a relatively small number of detergent monomers from membrane proteins. This process is accompanied by a relatively small and slow change in the FP anisotropy with respect to the initial value, rmax. The second phase depicts a large and fast alteration in the FP anisotropy, as it is representative of the loss of many detergent monomers from membrane proteins. This major change in the average molecular mass of proteomicelles is reflected in a drastic alteration of the FP anisotropy, and occurs in a single-exponential fashion (right-hand panels). The kinetic rates of both predesolvation and desolvation phases are dependent on the final detergent concentration (Wolfe et al., 2018a). For example, the kinetic rates are greater for lower detergent concentrations in the protein sample. Reprinted with permission from reference Wolfe et al. (2018b). Copyright 2018, American Chemical Society.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture>\u0000 </div>\u0000 </figure>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"97 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.81","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"137833480","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

N-Glycan Analysis by Ultra-Performance Liquid Chromatography and Capillary Gel Electrophoresis with Fluorescent Labeling n -聚糖的超高效液相色谱和荧光标记毛细管凝胶电泳分析

Current Protocols in Protein Science Pub Date : 2019-09-04 DOI: 10.1002/cpps.95

Maja Hanić, Gordan Lauc, Irena Trbojević-Akmačić

引用次数: 12

High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy 利用荧光偏振光谱高通量筛选蛋白质-洗涤剂配合物

Current Protocols in Protein Science Pub Date : 2019-08-30 DOI: 10.1002/cpps.96

Aaron J. Wolfe, Kyle J. Parella, Liviu Movileanu

{"title":"High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy","authors":"Aaron J. Wolfe, Kyle J. Parella, Liviu Movileanu","doi":"10.1002/cpps.96","DOIUrl":"10.1002/cpps.96","url":null,"abstract":"This article provides detailed protocols for a high-throughput fluorescence polarization (FP) spectroscopy approach to disentangle the interactions of membrane proteins with solubilizing detergents. Existing techniques for examining the membrane protein-detergent complex (PDC) interactions are low throughput and require high amounts of proteins. Here, we describe a 96-well analytical approach, which facilitates a scalable analysis of the PDC interactions at low-nanomolar concentrations of membrane proteins in native solutions. At detergent concentrations much greater than the equilibrium dissociation constant of the PDC, Kd, the FP anisotropy reaches a saturated value, so it is independent of the detergent concentration. On the contrary, at detergent concentrations comparable with or lower than the Kd, the FP anisotropy readout undergoes a time-dependent decrease, exhibiting a sensitive and specific detergent-dissociation signature. Our approach can also be used for determining the kinetic rate constants of association and dissociation. With further development, these protocols might be used in various arenas of membrane protein research that pertain to extraction, solubilization, and stabilization. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"97 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.96","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41906752","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: 0

Site- and Structure-Specific Quantitative N-Glycoproteomics Using RPLC-pentaHILIC Separation and the Intact N-Glycopeptide Search Engine GPSeeker 利用hplc - pentahilic分离和完整的n -糖肽搜索引擎GPSeeker进行位点和结构特异性定量n -糖蛋白组学研究

Current Protocols in Protein Science Pub Date : 2019-08-23 DOI: 10.1002/cpps.94

Kaijie Xiao, Zhixin Tian

引用次数: 13

G Protein–Coupled Estrogen Receptor Production Using an Escherichia coli Cell-Free Expression System 利用大肠杆菌无细胞表达系统制备G蛋白偶联雌激素受体

Current Protocols in Protein Science Pub Date : 2019-06-19 DOI: 10.1002/cpps.88

Samson A. Souza, Dane T. Kurohara, Chester L. Dabalos, Ho Leung Ng

引用次数: 2

Issue Information TOC 发布信息TOC

Current Protocols in Protein Science Pub Date : 2019-06-17 DOI: 10.1002/cpps.80

引用次数: 0

Proteome Analysis Using Gel-LC-MS/MS 使用凝胶- lc -MS/MS进行蛋白质组分析

Current Protocols in Protein Science Pub Date : 2019-06-10 DOI: 10.1002/cpps.93

Aaron R. Goldman, Lynn A. Beer, Hsin-Yao Tang, Peter Hembach, Delaine Zayas-Bazan, David W. Speicher

{"title":"Proteome Analysis Using Gel-LC-MS/MS","authors":"Aaron R. Goldman, Lynn A. Beer, Hsin-Yao Tang, Peter Hembach, Delaine Zayas-Bazan, David W. Speicher","doi":"10.1002/cpps.93","DOIUrl":"10.1002/cpps.93","url":null,"abstract":"This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed-phase nanocapillary ultra-high-pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC-MS/MS data are used to identify peptides and thereby infer proteins present in samples ranging from simple mixtures to very complex proteomes. Bottom-up proteome studies usually involve quantitative comparisons across several or many samples. For either situation, 1D SDS gels represent a simple, widely available technique that can be used to either fractionate complex proteomes or rapidly clean up low microgram samples with minimal losses. After gel separation and staining/destaining, appropriate gel slices are excised, and in-gel reduction, alkylation, and protease digestion are performed. Digests are then processed for LC-MS/MS analysis. Protocols are described for either sample fractionation with high-throughput processing of many samples or simple cleanup without fractionation. An optional strategy is to conduct in-solution reduction and alkylation prior to running gels, which is advantageous when a large number of samples will be separated into large numbers of fractions. Optimization of trypsin digestion parameters and comparison to in-solution protease digestion are also described. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"96 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.93","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40451343","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: 23

Comparing Complex Protein Samples Using Two-Dimensional Polyacrylamide Gels 使用二维聚丙烯酰胺凝胶比较复杂蛋白质样品

Current Protocols in Protein Science Pub Date : 2019-03-06 DOI: 10.1002/cpps.87

Sandra Harper, David W. Speicher

引用次数: 5

Experimental Assignment of Disulfide-Bonds in Purified Proteins 纯化蛋白中二硫键的实验分配

Current Protocols in Protein Science Pub Date : 2019-02-12 DOI: 10.1002/cpps.86

Hsin-Yao Tang, David W. Speicher

{"title":"Experimental Assignment of Disulfide-Bonds in Purified Proteins","authors":"Hsin-Yao Tang, David W. Speicher","doi":"10.1002/cpps.86","DOIUrl":"10.1002/cpps.86","url":null,"abstract":"The formation of disulfide bonds in proteins is an important post-translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified -SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence. These assignments provide insights into three-dimensional structure and contribute to the understanding of structural-functional relationships. This unit details a series of protocols that have been applied successfully to map disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Mass spectrometry is used to identify disulfide-containing peptides and determine sites of disulfide linkage. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"96 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.86","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36942182","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: 4

Tandem Affinity Purification and Mass Spectrometry (TAP-MS) for the Analysis of Protein Complexes 串联亲和纯化和质谱(TAP-MS)分析蛋白质复合物

Current Protocols in Protein Science Pub Date : 2019-02-01 DOI: 10.1002/cpps.84

Guillaume Adelmant, Brijesh K. Garg, Maria Tavares, Joseph D. Card, Jarrod A. Marto

引用次数: 16