CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, Liaoning, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Anal Bioanal Chem. 2019 Jul;411(18):4159-4166. doi: 10.1007/s00216-019-01770-w. Epub 2019 Apr 16.
Because of structural flexibility and acid lability, the identification of phosphorylated lysine (pLys) peptides is a great challenge. We report here a cleavable hydrophobic derivatization (CHD) strategy for the enrichment and identification of pLys peptides. First, 2,5-dioxopyrrolidin-1-yl-3-(decyldisulfanyl)propanoate was synthesized to react with dephosphorylated lysine peptides, and then the derived peptides were captured by a C column, followed by cleavage of the hydrophobic chain, with the specific label left on the target peptides for further identification. By CHD, the enrichment of pLys peptides from interfering peptides (1:1000 mass ratio) was achieved. Furthermore, CHD was applied to screen the pLys targets from Escherichia coli lysates, and 39 pLys sites from 35 proteins were identified. Gene Ontology (GO) analysis showed that these proteins played vital roles in catabolism, metabolism, biogenesis, and biosynthetic processes. All these results demonstrate that CHD might pave the way for comprehensive profiling of the pLys proteome.
由于结构的灵活性和酸的不稳定性，磷酸化赖氨酸（pLys）肽的鉴定是一个巨大的挑战。我们在这里报告了一种可裂解的疏水性衍生化（CHD）策略，用于富集和鉴定 pLys 肽。首先，合成了 2,5-二氧代吡咯烷-1-基-3-（癸基二硫基）丙酸盐，与去磷酸化的赖氨酸肽反应，然后将衍生肽通过 C 柱捕获，接着疏水性链被切断，目标肽上留下特定的标记，用于进一步鉴定。通过 CHD，从干扰肽（质量比为 1:1000）中富集 pLys 肽。此外，CHD 被应用于筛选大肠杆菌裂解物中的 pLys 靶标，从 35 种蛋白质中鉴定出 39 个 pLys 位点。基因本体论（GO）分析表明，这些蛋白质在分解代谢、代谢、生物发生和生物合成过程中发挥着重要作用。所有这些结果表明，CHD 可能为 pLys 蛋白质组的全面分析铺平道路。
