Determination of Lys-40 Acetylated Alpha-Tubulin in Rat Brain Tissue by Immuno Precipitation-Mass Spectrometry (IP-MS)

Abstract

Acetylation at ?-tubulin Lys-40 is a key post-translational modification (PTM) in the nervous system and is a potential biomarker for neurodegenerative diseases and xenobiotic-induced neurotoxicity. However, the absolute level of this key PTM has not been determined in the brain. Immunoprecipitation-mass spectrometry (IP-MS) combines antibody specificity and mass spectrometry selectivity and is being widely applied for the targeted quantitation of proteins within complex biomatrices. Lys-40 acetylation has not been previously studied with MS due to the lack of cleavage sites in the vicinity for trypsin and other common enzymes, making it challenging to identify a surrogate peptide for MS analysis. In this study, we report that such a peptide was identified for the first time after pepsin digestion, and an IP-MS-based assay was developed to absolutely quantitate Lys-40 acetylated ?-tubulin in rat brain tissue. The workflow includes pepsin digestion, immunoaffinity enrichment of the acetylated peptide, and quantitation using a stable isotope-labelled peptide. Only a small amount of brain tissue (2 mg) was required for each analysis. The method provided a linear range of 1.0–100.0 pmol/mg brain tissue, and selectivity, precision, and accuracy were validated. This method has been successfully applied in a preclinical study of chlorpyrifos neurotoxicity, and we observed a significant decrease of brain tubulin acetylation after chronic exposure to this organophosphate insecticide.

Keywords: Tubulin    acetylation,     immunoprecipitation, mass    spectrometry,  chlorpyrifos    and neurotoxicity