Reversed-phase high-performance liquid chromatographic separation of synthetic phosphopeptide isomers.
Reversed-phase high-performance liquid chromatographic separation of synthetic phosphopeptide isomers.
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合成磷酸肽异构体的反相高效液相色谱分离。
DOI：
10.1016/s0021-9673(01)89493-0
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Lee,VM
中科院分区：
文献类型：
--
作者：
OtvosJr,L;Tangoren,IA;Wroblewski,K;Hollosi,M;Lee,VM
通过反相高效液相色谱法分离由非磷酸化、二磷酸化和两种不同的单磷酸化异构体组成的混合物，选择性磷酸化的对应于人神经丝蛋白中等大小亚基的合成肽H-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly-Oh及其类似物。将该算法应用于4-9个氨基酸长的肽片段的预期保留时间，揭示了单磷酸化异构体的正确洗脱顺序。根据圆二色性研究，该洗脱顺序也与在键合相的表面上诱导构象取向的可能性相容。合成磷酸化反应的色谱分析表明，两种结构不同的单磷酸化肽的反应速率相似，这与体内位点定向反应相反。
Selectively phosphorylated synthetic peptides corresponding to the human neurofilament protein middle-sized subunit, H-Lys—Ser—Pro—Val—Pro—Lys—Ser—Pro—Val—Glu—Glu—Lys—Gly-Oh, and its analogues were separated by reversed-phase high-performance liquid chromatography of mixtures consisting of the non-phosphorylated, the diphosphorylated and the two different monophosphorylated isomers. Application of the algorithm for the expected retention times to 4–9 amino acid-long peptide fragments revealed the correct elution order of the monophosphorylated isomers. According to circular dichroism studies, this elution order is also compatible with the possibility of induced conformational orientation on the surface of the bonded phase. Chromatographic analysis of the synthetic phosphorylation reaction indicates that the reaction rates of the two structurally different monophosphorylated peptides are similar, which is in contrast to thein vivosite-directed reaction.