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

Xiangkun Yang, Sean X. Naughton, Zhen Han, Maomao He, Y. George Zheng, Alvin V Terry, Michael G. Bartlett

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2155-2163
Number of pages9
JournalAnalytical Chemistry
Volume90
Issue number3
DOIs
StatePublished - Feb 6 2018

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Acetylation
Pepsin A
Tubulin
Isotopes
Rats
Brain
Tissue
Peptides
Antibodies
Chlorpyrifos
Organophosphates
Biomarkers
Dilution
Mass spectrometry
Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

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. / Yang, Xiangkun; Naughton, Sean X.; Han, Zhen; He, Maomao; Zheng, Y. George; Terry, Alvin V; Bartlett, Michael G.

In: Analytical Chemistry, Vol. 90, No. 3, 06.02.2018, p. 2155-2163.

Research output: Contribution to journalArticle

Yang, Xiangkun ; Naughton, Sean X. ; Han, Zhen ; He, Maomao ; Zheng, Y. George ; Terry, Alvin V ; Bartlett, Michael G. / 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. In: Analytical Chemistry. 2018 ; Vol. 90, No. 3. pp. 2155-2163.
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