Single cell assay for protein palmitoylation dynamics

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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.