Site-Specific Identification of Lysine Acetylation Stoichiometries in Mammalian Cells

Functional
characterization of the lysine acetylation pathway requires
quantitative measurement of the modification abundance at the stoichiometry
level. Here, we developed a systematic workflow for global untargeted
identification of site-specific Lys acetylation stoichiometries in
mammalian cells. Our strategy includes an optimized protocol for in vitro chemical labeling of unmodified lysine with stable
isotope-encoded acetyl-NHS ester, deep proteomic profiling with a
high resolution mass spectrometer, and a new software tool for quantitative
analysis and stoichiometry determination. The workflow was validated
using in vitro chemically labeled BSA and synthetic
peptides with multiple Lys acetylations at various positions. In the
proof-of-concept study, we applied the strategy to analyze the proteome
of HeLa cells and determined the stoichiometries of over 600 acetylation
sites with good reproducibility. Sodium butyrate treatment induced
a significant increase of acetylation stoichiometries in HeLa cells.
Analysis of site-specific stoichiometry dynamics revealed the coregulation
of closely positioned acetylation sites on histones H3 and H4 upon
treatment.