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

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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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Abbreviations

HPDP-biotin:

N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide

LTQ:

Linear ion trap quadrupole

LC:

Liquid chromatography

EDTA:

Ethylenediaminetetraacetic acid

SDS:

Sodium dodecyl sulfate

NEM:

N-ethylmaleimide

PMSF:

Phenylmethanesulfonyl fluoride

TFA:

Trifluoroacetic acid

ESI:

Electrospray ionization

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Acknowledgments

This work was supported by the Scientific Research Fund of Zhejiang Provincial Education Department (No. Y200907062) and the National Key Basic Research Program of China (973 program) under grants 2004CB720008 and 2004CB720004.

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Authors and Affiliations

  1. Department of Basic Medical Sciences, Medical College of Taizhou University, Taizhou, Zhejiang, 318000, China

    Zhiqiang Zhao, Jianwei Deng & Xiaoming Wang

  2. Department of Nursing, Medical College of Taizhou University, Taizhou, Zhejiang, 318000, China

    Danfang Chen

  3. Proteomics Platform, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Zhiqiang Zhao, Junjie Hou, Zhensheng Xie & Fuquan Yang

  4. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Zhiqiang Zhao & Weimin Gong

Authors
  1. Zhiqiang Zhao
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  2. Junjie Hou
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  3. Zhensheng Xie
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  4. Jianwei Deng
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  5. Xiaoming Wang
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  6. Danfang Chen
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  7. Fuquan Yang
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Correspondence to Zhiqiang Zhao.

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Zhao, Z., Hou, J., Xie, Z. et al. Acyl-biotinyl Exchange Chemistry and Mass Spectrometry-Based Analysis of Palmitoylation Sites of In Vitro Palmitoylated Rat Brain Tubulin. Protein J 29, 531–537 (2010). https://doi.org/10.1007/s10930-010-9285-x

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