Diagonal Capillary Electrophoresis for Phosphorylation and Sialylation Analysis

修改摘要。这个研究小组最近发表了第一个描述对角线 毛细管电泳对角毛细管电泳是一种二维毛细管电泳 电泳在每个维度上采用相同的分离模式。的远端 第一个毛细管包含基于酶的微型反应器。分析物未被 反应器将在两个毛细管中具有相同的迁移时间，并将产生落在 重建的二维电泳图中的对角线。分析物经过 反应器中的酶改性在第二毛细管中具有不同的迁移时间 并且将产生从电泳图中的对角线上脱落的斑点。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。 电泳在这个建议中，我们将发展对角线毛细管电泳作为一个强大的 用于测定复杂样品磷酸化状态的工具。技术 与替代品相比具有显著的优势。最重要的是， 提供了修改程度的准确估计，而不会由于以下差异而产生伪影： 天然肽和修饰肽的电离效率。此外，该技术是完全自动化的： 一旦样品被加载到仪器中，就没有级分收集、移液或其它样品 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法更适合于分析 含有带电修饰的肽。

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.