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

Anal Chem. 2020 Aug 18;92(16):11204-11212. doi: 10.1021/acs.analchem.0c01683. Epub 2020 Jul 27.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Amino Acid Sequence
  • Animals
  • Humans
  • Ion Mobility Spectrometry
  • Lysine / chemistry
  • Mice, Inbred C57BL
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Processing, Post-Translational*
  • Proteomics / methods*
  • Stem Cells
  • Tubulin / analysis*
  • Tubulin / chemistry
  • Tubulin / metabolism

Substances

  • Tubulin
  • Lysine