期刊
PROTEIN JOURNAL
卷 29, 期 8, 页码 531-537出版社
SPRINGER
DOI: 10.1007/s10930-010-9285-x
关键词
Acyl-biotinyl exchange; Mass spectrometry; Tubulin; Palmitoylation site
资金
- Zhejiang Provincial Education Department [Y200907062]
- National Key Basic Research Program of China (973 program) [2004CB720008, 2004CB720004]
Research has shown that the palmitoyl group of alpha-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 alpha/beta tubulin heterodimer. C376 of alpha-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of alpha-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on alpha-tubulin was based on H-3-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 alpha/beta 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 alpha/beta tubulin heterodimer were palmitoylated.
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