Single cell assay for protein palmitoylation dynamics

描述（由申请人提供）：棕榈酰化是一种动态的、可逆的蛋白质翻译后修饰，被认为通过促进蛋白质在体内不同作用位点的运动和定位，在蛋白质信号传导中发挥作用。毛细管电泳（CE）为基础的单细胞测定将开发评估棕榈酰化荧光标记肽底物在体内。四种肽将被合成并荧光标记用作棕榈酰化底物：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，装载荧光标记肽的单细胞将被激光微移液系统裂解和取样，然后用MEKC-CE分离和定量。底物棕榈酰化将在酰基辅酶a、蓝蓝蛋白、2-溴棕榈酰酸盐和胰岛素的药理学刺激后进行评估。棕榈酰化动力学，前后药理学操作，也将评估每个标记底物。最后，荧光显微镜将用于关联底物位置棕榈酰化状态前后药理学操作。

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.