Acyl-biotinyl Exchange Chemistry and Mass Spectrometry-based Analysis of Palmitoylation Sites of in Vitro Palmitoylated Rat Brain Tubulin

Overview

Journal Protein J

Publisher Springer

Specialty Biochemistry

Date 2010 Oct 27

PMID 20976533

Citations 6

Authors

Zhiqiang Zhao

Junjie Hou

Zhensheng Xie

Jianwei Deng

Xiaoming Wang

Danfang Chen

Fuquan Yang

Weimin Gong

Affiliations

Soon will be listed here.

Abstract

Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.

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