Antibody-Free Targeted Proteomics Assay for Absolute Measurement of α-Tubulin Acetylation.
Anal Chem
; 92(16): 11204-11212, 2020 08 18.
Article
em En
| MEDLINE
| ID: mdl-32639142
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 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.
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Base de dados:
MEDLINE
Assunto principal:
Tubulina (Proteína)
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Processamento de Proteína Pós-Traducional
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Proteômica
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article