Diagonal Capillary Electrophoresis for Phosphorylation and Sialylation Analysis

描述（申请人提供）：本课题组近期首次发表对角毛细管电泳的描述。对角毛细管电泳是二维毛细管电泳的一种形式，在每个维度上采用相同的分离方式。第一毛细管的远端包含一个酶基微反应器。未经过反应器修饰的分析物在两根毛细血管中具有相同的迁移时间，并在重建的二维电泳图中产生落在对角线上的斑点。在反应器中进行酶修饰的分析物在第二毛细管中会有不同的迁移时间，并且会在电泳图中产生从对角线上脱落的斑点。去磷酸化和去硅基化导致负电荷的损失，这是微不足道的检测使用毛细管电泳。在本提案中，我们将开发对角毛细管电泳作为测定复杂样品的磷酸化和唾液酸状态的强大工具。与其他技术相比，该技术具有显著的优势。最重要的是，该技术提供了准确的估计修饰程度，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，该技术是全自动的：一旦样品被加载到仪器中，就不需要进行馏分收集、移液或其他样品操作。最后，该技术采用毛细管电泳，它似乎比反相液相色谱法更适合于分析含有带电修饰的肽。

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.