Acyl-biotinyl exchange chemistry and mass spectrometry-based analysis of palmitoylation sites of in vitro palmitoylated rat brain tubulin.
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.
Zhao
Zhiqiang
Z
Department of Basic Medical Sciences, Medical College of Taizhou University, Taizhou, Zhejiang 318000, China. xiaosazhaocn@yahoo.com.cn
Hou
Junjie
J
Xie
Zhensheng
Z
Deng
Jianwei
J
Wang
Xiaoming
X
Chen
Danfang
D
Yang
Fuquan
F
Gong
Weimin
W
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives; D001921:Brain / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D054878:Lipoylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000032:analysis / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D040901:Proteomics; D051381:Rats; D017421:Sequence Analysis; D015398:Signal Transduction; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D014404:Tubulin; C066114:biotin-streptavidin complex; D019308:Palmitic Acid; D001710:Biotin; D003545:Cysteine