Antibody-Free Targeted Proteomics Assay for Absolute Measurement of α‑Tubulin Acetylation

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 characterized
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 harboring K40 site. Using the synthetic unmodified and acetylated
stable isotope labeled 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 10–80% mixture of acetylated/nonacetylated
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.