title: A method to determine lysine acetylation stoichiometries.
abstract: Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodium butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA ; Bindley Bioscience Center, Discovery Park, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Ernesto S
authors.initials: ES
authors.lastname: Nakayasu
authors.affiliation: Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Si
authors.initials: S
authors.lastname: Wu
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Sydor
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Shukla
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Karl K
authors.initials: KK
authors.lastname: Weitz
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.affiliation: Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Kim K
authors.initials: KK
authors.lastname: Hixson
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA ; Institute for Basic Science, Seoul National University, Seoul 151-747, Republic of Korea.
authors.forename: Jong-Seo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Vladislav A
authors.initials: VA
authors.lastname: Petyuk
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Monroe
authors.affiliation: Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Ljiljiana
authors.initials: L
authors.lastname: Pasa-Tolic
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.affiliation: Biological Science Division and Environmental, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Ansong