QIMRBerghofer - Alpha-tubulin acetylation estimation

Antibody-free targeted proteomics assay for absolute measurement of α-tubulin acetylation

  • Organism: Homo sapiens, Mus musculus
  • Instrument: 6490 Triple Quadrupole LC/MS
  • SpikeIn: Yes
  • Keywords: α-tubulin acetylation
  • Lab head: Michelle Hill
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 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.
Experiment Description
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
Sample Description
1. Human olfactory neurosphere-derived stem (ONS) cell line (untreated and treated with Trichostatin A) AspN digest 2. Mouse brain homogenate and spinal cord
Created on 4/17/20, 7:12 AM
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicatesComment
Tubulin_Ova_AspN_SIS_Results_20200203_NoiRT_Linearity_Processed_Spinal cord_Brain homogenate.sky.zip2020-04-17 07:12:074253018019Linearity in brain homegenate and spinal cord along with unknown spinal cord estimation
Tubulin_Ova_AspN_SIS_Results_20200203_NoiRT_Linearity_Processed_Cell culture.sky.zip2020-04-17 07:12:074253018436Linearity and reproducibility in cell culture and Trichostatin A treatment of cells

Table S-1: List of MRM-MS assay transitions monitored.

Table S-2:

Sheet 1: Skyline export results for mouse brain homogenate and mouse spinal cord.

Sheet 2: Skyline export results for human olfactory neurosphere-derived stem (ONS) cell.

Sheet 3: Blank subtraction, normalisation, LOD estimation, LOQ estimation and % coefficient of variation calculation of the dataset.

Sheet 4: Calculations for SIL peptide injection along with results of unknown estimation and % acetylation estimation for the dataset.

  Attached Files  
   
 Table S-2.xlsx
 Table S-1.xlsx