Diagonal Capillary Electrophoresis for Phosphorylation and Sialylation Analysis

用于磷酸化和唾液酸化分析的对角毛细管电泳

• 批准号: 8710257
• 负责人: Norman J Dovichi
• 金额: $ 28.5万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710257
• 关键词: Affinity; Alkaline Phosphatase; Blood capillaries; Buffers; Capillary Electrophoresis; Charge; Chemistry; Collection; Complex; Computer software; Data; Data Analyses; Dimensions; Disease; Distal; Electrophoresis; Enzymes; Figs - dietary; Fluorescence; Glycopeptides; Hour; Imagery; Immune; Lasers; Lectin; Liquid Chromatography; Macrophage Activation; Mass Spectrum Analysis; Methods; Mitogen-Activated Protein Kinases; Modification; Morphologic artifacts; Peptides; Phase; Phosphopeptides; Phosphorylated Peptide; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Protein Dephosphorylation; Proteins; Publishing; Ras/Raf; Reagent; Research; Sampling; Spottings; System; Technology; Time; abstracting; base; capillary; design; detector; falls; innovation; instrument; ionization; mass spectrometer; migration; operation; protein function; sialylation; software development; tool; two-dimensional

Modified Abstract. This research group recently published the first description of diagonal capillary electrophoresis. Diagonal capillary electrophoresis is a form of two-dimensional capillary electrophoresis that employs identical separation modes in each dimension. The distal end of the first capillary incorporates an enzyme-based microreactor. Analytes that are not modified by the reactor will have identical migration times in the two capillaries and will generate spots that fall on the diagonal in a reconstructed two-dimensional electropherogram. Analytes that undergo enzymatic modification in the reactor will have a different migration time in the second capillary and will generate spots that fall off the diagonal in the electropherogram. Dephosphorylation and desialylation result in the loss of a negative charge, which is trivial to detect using capillary electrophoresis. In this proposal, we will develop diagonal capillary electrophoresis as a robust tool for the determination of the phosphorylation status of complex samples. The technology offers significant advantages compared with alternatives. Most importantly, the technology provides accurate estimates of the extent of modification without artifact due to differences in ionization efficiency of the native and modified peptides. Also, the technology is fully automated: once the sample is loaded into the instrument, no fraction collection, pipetting, or other sample manipulations are performed. Finally, the technology employs capillary electrophoresis, which appears to be better suited than reversed-phase liquid chromatography for the analysis of peptides containing charged modifications.

修改后的摘要。该研究小组最近发表了第一个对角线的描述 毛细管电泳。对角毛细管电泳是二维毛细管电泳的一种形式 在每个维度上采用相同分离模式的电泳。的远端 第一个毛细管包含一个基于酶的微反应器。未经过修饰的分析物 反应器将在两个毛细管中具有相同的迁移时间，并会产生落在 重建的二维电泳图中的对角线。经历过的分析物 反应器中的酶促修饰将在第二个毛细管中具有不同的迁移时间 并将产生偏离电泳图对角线的斑点。去磷酸化和 去唾液酸化会导致负电荷的损失，使用毛细管检测这是微不足道的 电泳。在本提案中，我们将开发对角毛细管电泳作为一种稳健的方法 用于确定复杂样品的磷酸化状态的工具。技术 与替代品相比具有显着的优势。最重要的是技术 提供对修改程度的准确估计，而不会由于差异而产生伪影 天然肽和修饰肽的电离效率。此外，该技术是完全自动化的： 将样品装入仪器后，无需进行馏分收集、移液或其他样品 进行操作。最后，该技术采用毛细管电泳， 似乎比反相液相色谱更适合分析 含有带电修饰的肽。