PT  - JOURNAL ARTICLE
AU  - Josue Baeza
AU  - Alexis J. Lawton
AU  - Jing Fan
AU  - Michael J. Smallegan
AU  - Ian Lienert
AU  - Tejas Gandhi
AU  - Oliver M. Bernhardt
AU  - Lukas Reiter
AU  - John M. Denu
TI  - Revealing dynamic protein acetylation across subcellular compartments
AID  - 10.1101/472530
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 472530
4099  - http://biorxiv.org/content/early/2019/08/15/472530.short
4100  - http://biorxiv.org/content/early/2019/08/15/472530.full
AB  - Protein acetylation is a widespread post-translational modification implicated in many cellular processes. Recent advances in mass spectrometry have enabled the cataloging of thousands of sites throughout the cell, however identifying regulatory acetylation marks have proven to be a daunting task. Knowledge of the kinetics and stoichiometry of site-specific acetylation are important factors to uncover function. Here, an improved method of quantifying acetylation stoichiometry was developed and validated, providing a detailed landscape of dynamic acetylation stoichiometry within cellular compartments. The dynamic nature of site-specific acetylation in response to serum stimulation was revealed. In two distinct human cell lines, growth factor stimulation led to site-specific, temporal acetylation changes, revealing diverse kinetic profiles that clustered into several groups. Overlap of dynamic acetylation sites among two different human cell lines suggested similar regulatory control points across major cellular pathways that include splicing, translation, and protein homeostasis. Rapid increases in acetylation on protein translational machinery suggest a positive regulatory role under pro-growth conditions. Lastly, higher median stoichiometry was observed in cellular compartments where active acetyltransferases are well-described.HighlightsDeveloped an integrated workflow for quantifying lysine acetylation stoichiometry and proteomic abundances.Identified site-specific acetylation stoichiometry distribution across cellular compartmentsRevealed rapid changes in protein acetylation that define large groups of sites and major pathwaysDetermined that highest acetylation stoichiometries are found in compartments with well-described acetyltransferase enzymes.In Brief Lysine acetylation is a widespread post-translational modification, but the dynamics of rapid changes in acetylation stoichiometry across cellular compartments are not known. Here, we developed an improved mass-spectrometry method to quantify the distribution and dynamic nature of acetylation stoichiometry upon growth factor stimulation. Nuclear proteins displayed the highest acetylation levels, and serum starvation-refeeding led to rapid acetylation changes, revealing major cellular pathways such as splicing, translation, and protein homeostasis are dynamically acetylated.ANOVAanalysis of varianceACNacetonitrileDDAdata-dependent acquisitionDIAdata-independent acquisitionDDTdithiothreitolGOgene ontologyHCDhigher energy collisional dissociationHPRPhigh pH reverse phaseIAAiodoacetamideKATlysine acetyltransferaseKDAClysine deacetylaseNCEnormalized collision energyPSMpeptide spectrum matchesQSSAquantitative site set functional score analysis