Single cell assay for protein palmitoylation dynamics

描述（由申请人提供）：棕榈酰化是蛋白质的动态和可逆的翻译后修饰，通过促进蛋白质在体内移动和定位到不同的作用位点，在蛋白质信号传导中发挥作用。将开发基于毛细管电泳（CE）的单细胞测定法，以评估体内荧光标记肽底物的棕榈酰化。将合成并荧光标记四种肽用作棕榈酰化底物：N-ras、H-ras、GAP-43和Gi-alpha 1。首先，这些底物的棕榈酰化和非棕榈酰化版本将通过胶束电动毛细管色谱法（MEKC）在体外分离。随后，使用激光微量移液管系统裂解负载有荧光标记肽的单细胞并取样，然后通过MEKC-CE进行分离和定量。在用酰基辅酶A、浅蓝菌素、2-溴棕榈酸酯和胰岛素进行药理学刺激后，评估底物棕榈酰化。还将评估每种标记底物在药理学操作前后的棕榈酰化动力学。最后，荧光显微镜将用于将底物位置与药理学操作前后的棕榈酰化状态相关联。

DESCRIPTION (provided by applicant): Palmitoylation, the dynamic and reversible posttranslational modification of proteins, is suggested to play a role in protein signaling by promoting movement and localization of proteins to different sites of action in vivo. A capillary electrophoresis (CE)-based single cell assay will be developed to assess palmitoylation of fluorescently-labeled peptide substrates in vivo. Four peptides will be synthesized and fluorescently-labeled for use as palmitoylation substrates: N-ras, H-ras, GAP-43, and Gi-alpha1. First, palmitoylated and non-palmitoylated versions of these substrates will be separated by micellar electrokinetic capillary chromatography (MEKC) in vitro. Subsequently, single cells loaded with fluorescently-labeled peptides will be lysed and sampled using a laser micropipette system, followed by separation and quantification by MEKC-CE. Substrate palmitoylation will be assessed following pharmacological stimulation with acyl-CoA, cerulenin, 2-bromopalmitate and insulin. Palmitoylation kinetics, before and after pharmacological manipulation, will also be assessed for each of the labeled substrates. Finally, fluorescence microscopy will be used to correlate substrate location to palmitoylation state before and after pharmacological manipulation.