Single cell assay for protein palmitoylation dynamics

描述(申请人提供)：棕榈酰化，蛋白质的动态和可逆的翻译后修饰，被认为通过促进蛋白质在体内不同作用部位的移动和定位而在蛋白质信号转导中发挥作用。建立了一种基于毛细管电泳法(CE)的单细胞分析方法，用于体内评价荧光标记的多肽底物的棕榈酰化反应。将合成四个肽并进行荧光标记，以用作棕榈酰化底物：N-ras、H-ras、GAP-43和Gi-Alpha1。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(MEKC)在体外分离。随后，装载有荧光标记的多肽的单个细胞将被裂解并使用激光微管系统采样，然后用MEKC-CE分离和定量。底物棕榈酰化将在用酰辅酶A、蓝菌素、2-溴棕榈酸酯和胰岛素进行药理刺激后进行评估。药物操作前后的棕榈酰化动力学也将对每一种标记底物进行评估。最后，将使用荧光显微镜将药物操作前后的底物位置与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.