Methodological Study On The Analysis Of Palmitoylated Proteomes Based On Acyl-biotinyl Exchange

Protein palmitoylation is a reversible post-translational modification that plays a key role in regulating protein-protein interactions,membrane localization,and intracellular signaling transduction.In addition,proteins such as GPCRs,transmembrane sodium/potassium pump,and voltage-gated sodium channel are regulated by palmitoylation in cardiac tissue.The studies of palmitoylated proteins by acyl-biotinyl exchange(ABE)have become increasingly mature,but mainly focuses on the nervous system,and little is known about the modifications in heart.Therefore,we used ABE method to label and identify palmitoylated proteins in heart,and further optimized the ABE method.Here,we labeled palmitoylated proteins in mouse cardiac tissue with Biotin-HPDP and 50 palmitoylated proteins were identified.Thereafter,by proteolysis of proteins from mouse heart tissue into polypeptides,we established an ABE method based on polypeptide to label palmitoylated proteins.In this method,we adsorbed the peptide sample onto the column,allowing the reaction buffer to slowly flow through the column and react with the peptide sample on the column.The purpose of removing residual reagents could be achieved by washing the column after each step of the reaction.This method simplifies the operation steps and greatly shortens the experiment time.The optimal experimental conditions in this study were that the sample was enzymatically hydrolyzed into peptides and free thiol was blocked with 10 mmol/L NEM buffer.The hydroxylamine(HA)reaction time was 90 min and the Biotin-maleimide working concentration was 10 ?mol/L.The sample should be lyophilized and enriched immediately after the ABE reaction.The optimal conditions for enrichment of antibiotin antibody were as follows: added 20 ?L of anti-biotin antibody agarose beads into 1 mg of ABE-labeled sample,and the palmitoylated proteins were enriched twice.This study further enriched the researches on protein palmitoylation modification and provided a reference for the optimization of ABE method.It has certain significance for the development of palmitoylation modification detection method.