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

基本信息

批准号：
8534195
负责人：
Norman J Dovichi
金额：
$ 27.5万
依托单位：
UNIVERSITY OF NOTRE DAME
依托单位国家：
美国
项目类别：
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/8534195
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

项目摘要

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.

修改的摘要。该研究小组最近发表了对角线的第一个描述毛细管电泳。对角毛细管电泳是二维毛细管的一种形式在每个维度中采用相同的分离模式的电泳。远端第一毛细管掺入了基于酶的微反应器。未经修改的分析物反应堆将在两个毛细管中具有相同的迁移时间，并且会产生落在的斑点重建的二维电池图中的对角线。经历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从电图中从对角线上掉落的斑点。去磷酸化和脱扬型导致损失负电荷，这很难检测到使用毛细管电泳。在此提案中，我们将开发对角线毛细管电泳作为强大的确定复杂样品磷酸化状态的工具。技术与替代方案相比，提供了显着的优势。最重要的是，技术由于差异的差异天然和改良肽的电离效率。此外，该技术是完全自动化的：一旦将样品加载到仪器中，就不会收集分数，移液或其他样品进行操作。最后，该技术采用毛细管电泳，这是似乎比相反的液体色谱更好地适合于分析含有带电修饰的肽。