DOI: 10.5061/dryad.3j9kd51sh
title: Mass spectrometry of axonemes from Tetrahymena thermophila CU428 and acetylation mutants

description: Acetylation of α-tubulin at the lysine 40 residue (αK40) by the
 ATAT1/MEC-17 acetyltransferase influences the properties of microtubules
 and is a widespread phenomenon in eukaryotic cells. Previous research
 indicates that microtubules that undergo acetylation at αK40 are more
 stable and resilient to damage. Notably, αK40 acetylation represents the
 sole identified post-translational modification site within the
 microtubule lumen, suggesting its role in regulating the lateral
 interactions among protofilaments within the microtubule structure. This
 investigation focuses on evaluating the impact of tubulin acetylation on
 doublet microtubules present in the cilia of Tetrahymena thermophila,
 employing mass spectrometry analysis. Cilia samples derived from
 Tetrahymena wild type, acetylation mutants (K40R and MEC17-Knockout), and
 non-acetylation mutants (RIB72B-Knockout and RIB72AB-Knockout) underwent
 comparative mass spectrometry analysis. The results from mass spectrometry
 revealed a correlation between αK40 acetylation and phosphorylation within
 the ciliary structures.
description: Purified axoneme from <em>Tetrahymena</em> cells
 underwent subsequent mass spectrometry examination. The axoneme solution
 was supplemented with Laemmli buffer at a 4X concentration (#1610747,
 Bio-Rad) to achieve a 1x concentration, and approximately 25-30 μg of
 protein was loaded onto an SDS‒PAGE gel. Electrophoresis was initiated but
 halted prior to the proteins traversing into the separation gel.
 Subsequently, a gel section containing all proteins within the sample was
 excised and subjected to in-gel digestion. The resulting peptides (~2 μg)
 were chromatographically separated utilizing a Dionex Ultimate 3000 UHPLC
 system. Initially, peptides were loaded onto a Thermo Acclaim Pepmap
 precolumn (Thermo, 75 μm ID × 2 cm with 3 μm C18 beads) and subsequently
 onto an Acclaim Pepmap Easyspray analytical column (Thermo, 75 μm × 25 cm
 with 2 μm C18 beads), with separation achieved at a flow rate of 200
 nl/min employing a gradient of 2-35% solvent (acetonitrile containing 0.1%
 formic acid) over 2 hours. Peptides possessing a charge of 2+ or higher
 were detected utilizing a Thermo Orbitrap Fusion mass spectrometer
 operating at a resolution of 120,000 (FWHM in MS1, 15,000 for MS/MS). The
 acquired data were interrogated against the <em>Tetrahymena
 thermophila </em>protein dataset sourced from UniProt (https://www.uniprot.org/). The subsequent analysis of the mass spectrometry data was conducted using Scaffold_4.8.4 (Proteome Software Inc.). Proteins with a mean value of exclusive unique peptide count of 2 or greater in the wildtype mass spectrometry outcomes were selected for further analysis. Additionally, raw mass spectrometry data were normalized by the total spectra.
description: # Mass spectrometry of axonemes from *Tetrahymena thermophila* CU428 and
 acetylation mutants
 [https://doi.org/10.5061/dryad.3j9kd51sh](https://doi.org/10.5061/dryad.3j9kd51sh) Mass spectrometry of the axoneme (no ciliary membrane) from *Tetrahymena thermophila WT* (CU428 strain), *K40R* (alpha-tubulin substitution of lysine 40 with arginine), *MEC17-KO* (Knockout of alphaTAT1/MEC17), *RIB72B-KO* (knockout of the gene encoding microtubule inner protein RIB72B) and *RIB72A/B-KO* (double knockout of the genes encoding microtubule inner proteins *RIB72A* and *RIB72B*) mutants. ## Description of the data and file structure We submitted our mass spectrometry data after searching against the *T. thermophila* protein dataset *f*rom UniProt ([https://www.uniprot.org/)](https://www.uniprot.org/\)). The file cu428_k40r_mec17_rib72ab_merged.sf3 file can be downloaded and opened with the Free version of Scaffold Viewer. CU428: Wild type (with triplicate CU428_Sample_1, CU428_Sample_2, CU428_Sample_3) K40R: alpha-tubulin substitution of lysine 40 with arginine (with triplicate K40R_Sample_1, K40R_Sample_2, K40R_Sample_3) MEC17KO: Knockout of alphaTAT1/MEC17 (with triplicate MEC17KO_Sample_1, MEC17KO_Sample_2, MEC17KO_Sample_3) RIB72BKO: Knockout of the gene encoding microtubule inner protein RIB72B (with triplicate RIB72BKO_Sample_1, RIB72BKO_Sample_2, RIB72BKO_Sample_3) RIB72ABKO: Knockout of the genes encoding microtubule inner protein RIB72A and RIB72B (with triplicate RIB72ABKO_Sample_1, RIB72ABKO_Sample_2, RIB72ABKO_Sample_3) ## Sharing/Access information Data was derived from the following sources: * Part of the mass spectrometry data (K40R and MEC17-KO mutant) was deposited earlier in another dataset [https://datadryad.org/stash/dataset/doi:10.5061/dryad.p2ngf1vqv](https://datadryad.org/stash/dataset/doi:10.5061/dryad.p2ngf1vqv)