期刊
JOURNAL OF PROTEOME RESEARCH
卷 11, 期 3, 页码 1913-1923出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr2011044
关键词
AGBL2; detyrosination; glutamylation; tandem hybrid mass spectrometry; tubulin methylation; microtubule targetting drugs (MTTDs); nano-LC; proteinase K; tubulin-alpha; tubulin C-terminal tail
资金
- NIH [R01CA129813, 1 P01 CA130821]
- Charles and Mary Latham Fund
- Comprehensive Cancer Center Core Facilities (NIH) [P30 CA51008]
Tubulin-alpha(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-alpha(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-alpha(1A/1B) glutamylation was found exclusively on the E-445 residue. We here describe the analysis of tubulin-alpha(1A/1B) glutamylation and detyrosination after 2-DE separation, trypsin and proteinase K in-gel digestion, and nanoUPLC-ESI-QqT0E-MS/MS of mouse brain and Tyrosinated, detyrosinated, and Delta 2-tubulin-alpha(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 E-445, glutamylation on mouse and bovine tubulin-alpha(1A/1B) CTTs was identified on E-441 and E-443 with MASCOT Expect values below 0.01. O-Methylation of glutamates was also observed.
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