Analysis of Tubulin Alpha-1A/1B C-Terminal Tail Post-translational Poly-glutamylation Reveals Novel Modification Sites

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• 作者：Ziad J. Sahab ; Alexander Kirilyuk ; Lihua Zhang ; Zahraa I. Khamis ; Petr Pompach ; YouMe Sung ; Stephen W. Byers
• 刊名：Journal of Proteome Research
• 出版年：2012
• 出版时间：March 2, 2012
• 年：2012
• 卷：11
• 期：3
• 页码：1913-1923
• 全文大小：726K
• 年卷期：v.11,no.3(March 2, 2012)
• ISSN：1535-3907

文摘

Tubulin-伪_1A/1B C-terminal tail (CTT) has seven glutamic acid residues among the last 11 amino acids of its sequence that are potential sites for glutamylation. Cleavage of C-terminal tyrosine resulting in the detyrosinated form of tubulin-伪_1A/1B is another major modification. These modifications among others bring about highly heterogeneous tubulin samples in brain cells and microtubules, play a major role in directing intracellular trafficking, microtubule dynamics, and mitotic events, and can vary depending on the cell and disease state, such as cancer and neurodegenerative disorders. Identified previously using primary mass spectrometry (MS) ions and partial Edman sequencing, tubulin-伪_1A/1B glutamylation was found exclusively on the E⁴⁴⁵ residue. We here describe the analysis of tubulin-伪_1A/1B glutamylation and detyrosination after 2-DE separation, trypsin and proteinase K in-gel digestion, and nanoUPLC-ESI-QqTOF-MS/MS of mouse brain and bovine microtubules. Tyrosinated, detyrosinated, and 螖2-tubulin-伪_1A/1B CTTs were identified on the basis of a comparison of fragmentation patterns and retention times between endogenous and synthetic peptides. Stringent acceptance criteria were adapted for the identification of novel glutamylation sites. In addition to the previously identified site at E⁴⁴⁵, glutamylation on mouse and bovine tubulin-伪_1A/1B CTTs was identified on E⁴⁴¹ and E⁴⁴³ with MASCOT Expect values below 0.01. O-Methylation of glutamates was also observed.

Keywords:

AGBL2; detyrosination; glutamylation; tandem hybrid mass spectrometry; tubulin methylation; microtubule targetting drugs (MTTDs); nano-LC; proteinase K; tubulin-alpha; tubulin C-terminal tail