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: 8024455
• 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/8024455
• 关键词: Affinity; Alkaline Phosphatase; Blood capillaries; Buffers; Capillary Electrophoresis; Cell Line; Charge; Chemistry; Collection; Complex; Computer software; Data; Data Collection; Dimensions; Disease; Distal; Electrophoresis; Enzymes; Excision; Figs - dietary; Fluorescence; Glycopeptides; Hour; Imagery; Immune; Lasers; Lectin; Liquid Chromatography; Mass Spectrum Analysis; Methods; Mitogen-Activated Protein Kinases; Modification; Morphologic artifacts; Neuraminidase; Oligosaccharides; Peptides; Phase; Phosphopeptides; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Protein Dephosphorylation; Proteins; Publishing; Ras/Raf; Reagent; Research; Sampling; Sialic Acids; Site; Spottings; System; Technology; Time; base; capillary; design; detector; falls; glycosylation; innovation; instrument; ionization; macrophage; mass spectrometer; migration; operation; protein function; sialylation; sugar; tool; two-dimensional

DESCRIPTION (provided by applicant): 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 and sialic acid 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. PUBLIC HEALTH RELEVANCE: The phosphorylation and glycosylation status of a protein modulates the activity of that protein, which is important in a wide range of diseases. This proposal provides the first fully automated method to identify phosphorylated and specific glycosylated peptides without interference.

描述（由申请人提供）：该研究小组最近发表了对角毛细管电泳的第一个描述。对角毛细管电泳是二维毛细管电泳的一种形式，在每个维度中采用相同的分离模式。第一个毛细管的远端结合了基于酶的微反应器。反应器未修改的分析物将在两个毛细血管中具有相同的迁移时间，并且会在重建的二维电池图中产生落在对角线上的斑点。在第二毛细管中进行酶促修饰的分析物将具有不同的迁移时间，并且会产生斑点，这些斑点会从电要图中的对角线上掉落。去磷酸化和脱酰化导致负电荷的损失，这对于使用毛细管电泳检测很微不足道。 在此提案中，我们将开发对角线毛细管电泳，作为确定复杂样品的磷酸化和唾液酸状态的强大工具。与替代方案相比，该技术具有显着优势。最重要的是，由于天然和修饰肽的电离效率差异，该技术在没有伪像的情况下进行了准确的修饰程度估计。另外，该技术是完全自动化的：一旦将样品加载到仪器中，就不会执行分数收集，移液或其他样品操作。最后，该技术采用了毛细管电泳，与反相液相色谱法相比，该技术似乎更适合用于分析含有带电修饰的肽。 公共卫生相关性：蛋白质的磷酸化和糖基化状态调节该蛋白质的活性，这在广泛的疾病中很重要。该建议提供了第一种完全自动化的方法，可以识别而不会干扰的磷酸化和特定的糖基化肽。