Diagonal Capillary Electrophoresis for Phosphorylation and Sialylation Analysis

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，其在每个维度上采用相同的分离模式。第一毛细管的远端结合了基于酶的微反应器。未被反应器改性的分析物在两个毛细管中具有相同的迁移时间，并将在重建的二维电泳图中产生落在对角线上的斑点。在反应器中经历酶改性的分析物在第二毛细管中将具有不同的迁移时间，并且将产生在电泳图中偏离对角线的斑点。去磷酸化和去唾液酸化导致负电荷的损失，这对于使用毛细管电泳检测是微不足道的。 在这项提案中，我们将开发对角线毛细管电泳作为一个强大的工具，用于确定复杂样品的磷酸化和唾液酸状态。与替代品相比，该技术具有显著的优势。最重要的是，该技术提供了修饰程度的准确估计，而不会由于天然肽和修饰肽的电离效率差异而产生伪影。此外，该技术是完全自动化的：一旦样品被加载到仪器中，就不进行馏分收集、移液或其他样品操作。最后，该技术采用毛细管电泳，这似乎是更好地适合于分析含有带电修饰的肽比反相液相色谱。 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，这在广泛的疾病中是重要的。该提案提供了第一个完全自动化的方法来识别磷酸化和特定的糖基化肽，而不受干扰。

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.