3 years ago

Mass Spectrometric Quantitation of Tubulin Acetylation from Pepsin-Digested Rat Brain Tissue Using a Novel Stable-Isotope Standard and Capture by Anti-Peptide Antibody (SISCAPA) Method

Mass Spectrometric Quantitation of Tubulin Acetylation
from Pepsin-Digested Rat Brain Tissue Using a Novel Stable-Isotope
Standard and Capture by Anti-Peptide Antibody (SISCAPA) Method
Zhen Han, Sean X. Naughton, Alvin V. Terry, Michael G. Bartlett, Y. George Zheng, Maomao He, Xiangkun Yang
Acetylation of α-tubulin at Lys-40 is a potential biomarker for cognitive deficits in various neurological disorders. However, this key post-translational modification (PTM) has not been previously studied with mass spectrometry, due to the inadequate distribution of tryptic cleavage sites. Following peptic digestion, a surrogate sequence containing this key PTM site was identified and was found to be stable and quantitatively reproducible. A highly sensitive and specific SISCAPA–LC–MS method for quantitating rat brain tubulin acetylation was developed, validated, and applied, and only required a small amount of tissue (2.2 mg). This workflow includes peptic digestion, stable-isotope dilution, capture with antiacetylated peptide antibody bound on protein G beads, and quantitation using LC–MS. The method allowed a lower limit of quantitation at 2.50 pmol/mg and provided a linear range of 2.50–62.50 pmol/mg. Selectivity, intra and interday precision and accuracy were also validated. This method has been successfully applied in a preclinical study of organophosphate neurotoxicity, and we found that chronic exposure to chlorpyrifos led to a significant and persistent inhibition of brain tubulin acetylation.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b04484

DOI: 10.1021/acs.analchem.7b04484

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