Test for potential biomarker established

Relevance for HSP clinical trials

A test (assay) for a biomarker of microtubule stability that has potential for use in the HSP clinical trials currently being planned, has been successfully developed. A collaboration between members of the HSP Research Program team and the Precision and Systems Biomedicine group at the Queensland Institute of Medical Research (QIMR) resulted in the breakthrough. Gautam Wali in collaboration with Alok Shah at QIMR spearheaded the effort funded by this Foundation and the SP Foundation in the USA.

No assay existed for measuring levels of the potential biomarker compound in mouse tissue samples in the recent drug dose level studies. As has happened on several other occasions in the HSP Research Program over the years, a reliable and valid assay needed to be developed from scratch. These new assays have been vitally important to the success of the HSP Research Program over the years.


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.

SOURCE: Anal Chem. 2020 Aug 18;92(16):11204-11212. doi: 10.1021/acs.analchem.0c01683. Epub 2020 Jul 27. PMID: 32639142

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

Alok K Shah 1Gautam Wali 2Carolyn M Sue 2Alan Mackay-Sim 2 3Michelle M Hill 1 4

1 QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Queensland 4006, Australia.

2 Department of Neurogenetics, Kolling Institute, Sydney Medical School, University of Sydney, Sydney, New South Wales 2065, Australia.

3 Griffith Institute for Drug Discovery, Griffith University, 46 Don Young Rd, Nathan, Queensland 4111, Australia.

4 Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Herston, Queensland 4006, Australia.

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