title: Analysis of tubulin alpha-1A/1B C-terminal tail post-translational poly-glutamylation reveals novel modification sites.
abstract: Tubulin-α(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-α(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-α(1A/1B) glutamylation was found exclusively on the E(445) residue. We here describe the analysis of tubulin-α(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 Δ2-tubulin-α(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-α(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.
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University , Washington, DC 20007, United States. zjs3@georgetown.edu
authors.forename: Ziad J
authors.initials: ZJ
authors.lastname: Sahab
authors.forename: Alexander
authors.initials: A
authors.lastname: Kirilyuk
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zahraa I
authors.initials: ZI
authors.lastname: Khamis
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Youme
authors.initials: Y
authors.lastname: Sung
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Byers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D053719:Tandem Mass Spectrometry; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D020033:Protein Isoforms; C569741:Tuba1a protein, mouse; D014404:Tubulin; D018698:Glutamic Acid; D014443:Tyrosine