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BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation
Journal of Proteome Research ( IF 4.4 ) Pub Date : 2017-12-28 00:00:00 , DOI: 10.1021/acs.jproteome.7b00775 Dae In Kim 1 , Jevon A. Cutler 1, 2, 3 , Chan Hyun Na 1, 4 , Sina Reckel 5 , Santosh Renuse 1, 4 , Anil K. Madugundu 1, 6 , Raiha Tahir 3, 7 , Hana L. Goldschmidt 8 , Karen L. Reddy 3, 9 , Richard L. Huganir 8 , Xinyan Wu 1, 3 , Natasha E. Zachara 3 , Oliver Hantschel 5 , Akhilesh Pandey 1, 3, 4, 10
Journal of Proteome Research ( IF 4.4 ) Pub Date : 2017-12-28 00:00:00 , DOI: 10.1021/acs.jproteome.7b00775 Dae In Kim 1 , Jevon A. Cutler 1, 2, 3 , Chan Hyun Na 1, 4 , Sina Reckel 5 , Santosh Renuse 1, 4 , Anil K. Madugundu 1, 6 , Raiha Tahir 3, 7 , Hana L. Goldschmidt 8 , Karen L. Reddy 3, 9 , Richard L. Huganir 8 , Xinyan Wu 1, 3 , Natasha E. Zachara 3 , Oliver Hantschel 5 , Akhilesh Pandey 1, 3, 4, 10
Affiliation
Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC–MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.
中文翻译:
BioSITe:直接检测和定量特定部位生物素化的方法
基于生物素的标记策略已广泛用于研究蛋白质间相互作用,亚细胞蛋白质组和翻译后修饰,以及用于药物发现。尽管抗生蛋白链菌素对生物素的高亲和力极大地促进了生物素化蛋白的捕获,但如目前所采用的那样,它仍然对生物素化肽的回收提出了挑战。在这里,我们描述了一种称为生物素化位点识别技术(BioSITe)的策略,用于捕获用于LC-MS / MS分析的生物素化肽。我们证明了将BioSITe应用于邻近依赖性标记方法,APEX和BioID以及用于识别O-GlcNAc修饰位点的基于生物素的点击化学策略时的实用性。我们展示了同位素标记生物素在定量BioSITe实验中的应用,该实验简化了差异相互作用组分析并消除了对诸如SILAC之类的代谢标记策略的需求。我们的数据还凸显了位点特异性生物素化在提供有关蛋白质和蛋白质复合物的空间和拓扑信息方面的潜在价值。总体而言,我们预计在检测生物素化位点很重要的研究中,BioSITe将取代传统方法。
更新日期:2017-12-29
中文翻译:
BioSITe:直接检测和定量特定部位生物素化的方法
基于生物素的标记策略已广泛用于研究蛋白质间相互作用,亚细胞蛋白质组和翻译后修饰,以及用于药物发现。尽管抗生蛋白链菌素对生物素的高亲和力极大地促进了生物素化蛋白的捕获,但如目前所采用的那样,它仍然对生物素化肽的回收提出了挑战。在这里,我们描述了一种称为生物素化位点识别技术(BioSITe)的策略,用于捕获用于LC-MS / MS分析的生物素化肽。我们证明了将BioSITe应用于邻近依赖性标记方法,APEX和BioID以及用于识别O-GlcNAc修饰位点的基于生物素的点击化学策略时的实用性。我们展示了同位素标记生物素在定量BioSITe实验中的应用,该实验简化了差异相互作用组分析并消除了对诸如SILAC之类的代谢标记策略的需求。我们的数据还凸显了位点特异性生物素化在提供有关蛋白质和蛋白质复合物的空间和拓扑信息方面的潜在价值。总体而言,我们预计在检测生物素化位点很重要的研究中,BioSITe将取代传统方法。