Acetylation of α-tubulin at conserved lysine 40 (K40) amino acid residue regulates microtubule dynamics and controls a wide range of cellular activities. Dysregulated microtubule dynamics characterised by differential α-tubulin acetylation is a hallmark of cancer, neurodegeneration and other complex disorders. Hence, accurate quantitation of α-tubulin acetylation is required in human disease and animal model studies. We developed a novel antibody-free proteomics assay to measure α-tubulin acetylation targeting protease AspN-generated peptides harbouring K40 site. Using the synthetic unmodified and acetylated stable isotope labelled peptides DKTIGGG and DKTIGGGD, we demonstrate assay linearity across 4 log magnitude and reproducibility of <10% coefficient of variation . The assay accuracy was validated by titration of 10% to 80% mixture of acetylated/non-acetylated α-tubulin peptides in the background of human olfactory neurosphere-derived stem (ONS) cell matrix. Furthermore, in agreement with antibody-based high content microscopy analysis, the targeted proteomics assay reported an induction of α-tubulin K40 acetylation upon Trichostatin A stimulation of ONS cells. Independently, we found 35.99% and 16.11% α-tubulin acetylation for mouse spinal cord and brain homogenate tissue, respectively, as measured by our assay. In conclusion, this simple, antibody-free proteomics assay enables quantitation of α-tubulin acetylation, and is applicable across various fields of biology and medicine.

It includes following experiments: 1. Linearity and reproducibility assessment of unmodified and acetylated alpha-tubulin SIL peptides 2. Linearity assessment to estimate various levels of alpha-tubulin acetylation with spiked-in SIL peptides 3. Effect of Trichostatin A on tubulin acetylation 4. Assessment of alpha-tubulin acetylation in mouse tissue

1. Human olfactory neurosphere-derived stem (ONS) cell line (untreated and treated with Trichostatin A) AspN digest 2. Mouse brain homogenate and spinal cord