Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide

Abstract

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly-palmitoylated GAPpeptide product was confirmed by mass spectroscopy. The GAP-peptide substrate was separated from the palmitoylated peptide product in under 7 minutes by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.

Protein Lipidation: Occurrence, Mechanisms, Biological Functions, and Enabling Technologies

“…Difference gel electrophoresis based proteomics 705 detects slight differences in pI values and the relative mobility of palmitoylated and depalmitoylated proteinsMicellar electrokinetic chromatography was used identify GAP-43 palmitoylation in vitro. 706 The separation of palmitoylated and unmodified GAP-43 peptides can be performed in less than 7 min. Gas chromatography-MS and liquid chromatography-MS methods have also been developed to directly identify and quantify palmitoylation and other lipidations by comparing the retention time and mass spectrum with standard samples.…”

“…Difference gel electrophoresis based proteomics detects slight differences in p I values and the relative mobility of palmitoylated and depalmitoylated proteins. Micellar electrokinetic chromatography was used identify GAP-43 palmitoylation in vitro . The separation of palmitoylated and unmodified GAP-43 peptides can be performed in less than 7 min.…”

Micellar electrokinetic chromatography: A practical overview of current methodological and instrumental advances

“…Most of the LIF–MEKC methods reported in the period covered by this review use visible lasers as the excitation source due to the wide availability of label agents that are excitable by this kind of laser. Concretely, the commercial argon‐ion laser excitation source has been consolidated and therefore used in many LIF–MEKC applications 53, 54, 143–150; few alternatives have been proposed 19, 116, with diode laser being the most interesting alternative one 36, 151–153. A large number of applications have been reported over the period covered in this review; as in the previous ones reviews 1–3, many of them involving the determination of analytes possessing no native fluorescence.…”

MEKC as a powerful growing analytical technique

“…The extended application of MEKC in cancer research has been reported in several publications 213, 286–290. MEKC has been used to explore different aspects of cancer research from the search for biological markers in serum, urine, and tissue, to epidemiological studies, to the exploration of anticancer drugs in plant and marine organisms 291.…”