Mass spectrometry of axonemes from Tetrahymena thermophila CU428 and acetylation mutants

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 correlat..., Purified axoneme from Tetrahymena 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...., , # 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_Sa...