Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn
Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn
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DOI：
10.2116/analsci.20.1313
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发表时间：
2004-09-01
影响因子：
1.6
通讯作者：
Furuno, M
中科院分区：
文献类型：
--
作者：
Kuroda, I;Shintani, Y;Furuno, M
提出了一种利用二氧化钛作为柱前介质的磷酸肽选择性分析与柱切换HPLC相结合的方法。磷酸肽选择性地富集在二氧化钛包装内的蛋白质/肽混合物，没有任何额外的程序，并通过柱切换高效液相色谱法进行分析。首先，通过在酸性条件下在二氧化钛填料上捕集磷化合物来从复杂混合物中分离磷化合物，其中非磷酸化化合物流出预柱。随后，磷化合物在特定条件下从二氧化钛柱中解吸并分析。磷化合物在二氧化钛表面上的行为，特别是吸附/脱附，被精确地检查和优化。磷酸缓冲液连续用于洗脱磷酸肽的二氧化钛表面上的自由磷酸基团的竞争。从成功的选择性浓缩/分析磷酸肽与柱切换HPLC与二氧化钛预柱，一种新的磷酸肽选择性反相高效液相色谱分析已被证明有应用的可能性作为磷酸蛋白质组学的工具。
A methodology of phosphopeptide-selective analysis coupled with column-switching HPLC utilizing titania as precolumn media is presented. Phosphopeptides were selectively enriched on titania packing within a protein/peptide mixture without any additional procedure, and analyzed by column-switching high-performance liquid chromatography. First, phospho-compounds were separated from complex mixtures by trapping them under acidic conditions on a titania packing, where non-phosphorylated compounds were effused out of the precolumn. Subsequently, phospho-compounds were desorbed from the titania column under a specific condition and analyzed. The behavior of phospho-compounds on a titania surface, especially adsorption/desorption, was precisely examined and optimized. A phosphoric buffer was successively employed for the elution of phosphopeptides on a titania surface by competition with the free phosphate group. From the successes of a selective concentration/analysis of phosphopeptides with column-switching HPLC with a titania precolumn, a novel phosphopeptide-selective RP-HPLC analysis has been shown to have an application possibility as a tool for phosphoproteomics.