Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis
Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis
复制标题
DOI：
10.1016/j.ab.2021.114247
复制
发表时间：
2021-06-09
影响因子：
2.9
通讯作者：
Nohta, Hitoshi
中科院分区：
文献类型：
--
作者：
Kawasue, Shimba;Sakaguchi, Yohei;Nohta, Hitoshi
蛋白质的翻译后修饰涉及蛋白质功能和高阶结构。在这些修饰中，磷酸化是重要的细胞内信号转导途径。已经开展了许多使用液相色谱-电喷雾电离-质谱（LCESI-MS）分析磷酸化蛋白质的研究。然而，由于高度磷酸化蛋白质的处理困难，对其分析的报道很少。因此，我们开发了一种分析方法，将肽中包含的多个磷酸基团转化为全氟烷基，以便使用氟亲和力进行选择性分析。此处，胰蛋白酶消化的 β-酪蛋白片段肽 [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR 和 FQ(pSer) EEQQQTEDELQDK] 用作模型磷酸化肽。 1H,1H,2H,2H-全氟辛硫醇（PFOT）和2,2,2-三氟乙硫醇（TFET）分别用作单磷酸化肽和多磷酸化肽的衍生试剂，通过β-消除/迈克尔加成进行衍生化。通过LC-ESI-MS分析衍生物。氟液相色谱柱通常用于选择性保留氟衍生肽，预期将其与污染物和非磷酸化肽分离。当将该方法应用于 β-酪蛋白时，TFET 和 PFOT 衍生的肽被牢固地保留在荧光 LC 柱中，并在色谱图上与非磷酸化的肽清楚地分离。因此，所开发的方法能够定量单磷酸化和多磷酸化肽，并且适合在蛋白质组学中应用。
Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LCESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested beta-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer) EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via beta-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to beta-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics.