skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A Method to determine lysine acetylation stoichiometries

Abstract

A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. 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 lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1171295
Report Number(s):
PNNL-SA-96571
42294; 400412000
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: International Journal of Proteomics, 2014:Article No. 730725
Country of Publication:
United States
Language:
English
Subject:
Environmental Molecular Sciences Laboratory

Citation Formats

Nakayasu, Ernesto S., Wu, Si, Sydor, Michael A., Shukla, Anil K., Weitz, Karl K., Moore, Ronald J., Hixson, Kim K., Kim, Jong Seo, Petyuk, Vladislav A., Monroe, Matthew E., Pasa-Tolic, Ljiljana, Qian, Weijun, Smith, Richard D., Adkins, Joshua N., and Ansong, Charles. A Method to determine lysine acetylation stoichiometries. United States: N. p., 2014. Web. doi:10.1155/2014/730725.
Nakayasu, Ernesto S., Wu, Si, Sydor, Michael A., Shukla, Anil K., Weitz, Karl K., Moore, Ronald J., Hixson, Kim K., Kim, Jong Seo, Petyuk, Vladislav A., Monroe, Matthew E., Pasa-Tolic, Ljiljana, Qian, Weijun, Smith, Richard D., Adkins, Joshua N., & Ansong, Charles. A Method to determine lysine acetylation stoichiometries. United States. doi:10.1155/2014/730725.
Nakayasu, Ernesto S., Wu, Si, Sydor, Michael A., Shukla, Anil K., Weitz, Karl K., Moore, Ronald J., Hixson, Kim K., Kim, Jong Seo, Petyuk, Vladislav A., Monroe, Matthew E., Pasa-Tolic, Ljiljana, Qian, Weijun, Smith, Richard D., Adkins, Joshua N., and Ansong, Charles. Mon . "A Method to determine lysine acetylation stoichiometries". United States. doi:10.1155/2014/730725.
@article{osti_1171295,
title = {A Method to determine lysine acetylation stoichiometries},
author = {Nakayasu, Ernesto S. and Wu, Si and Sydor, Michael A. and Shukla, Anil K. and Weitz, Karl K. and Moore, Ronald J. and Hixson, Kim K. and Kim, Jong Seo and Petyuk, Vladislav A. and Monroe, Matthew E. and Pasa-Tolic, Ljiljana and Qian, Weijun and Smith, Richard D. and Adkins, Joshua N. and Ansong, Charles},
abstractNote = {A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. 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 lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.},
doi = {10.1155/2014/730725},
journal = {International Journal of Proteomics, 2014:Article No. 730725},
number = ,
volume = ,
place = {United States},
year = {Mon Jul 21 00:00:00 EDT 2014},
month = {Mon Jul 21 00:00:00 EDT 2014}
}
  • 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 sodiummore » 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.« less
  • Histone acetylation plays important roles for the regulation of many fundamental cellular processes. Saccharomyces cerevisiae Rtt109 is an important class of histone acetyltransferases (HATs), which promote genome stability by directly acetylating newly synthesized histone H3 lysine 56 (H3-K56) through an unknown mechanism. Here, we report the crystal structures of Rtt109 at 2.2 A and Rtt109/Acetyl-CoA binary complex at 1.9 A. The structure displays a vise-like topology with mixed three-layered ?/? module forming the central module, whose core region resembles the structure of GCN5 HAT domain and P300/CBP HAT domain. Using structural and biochemical analyses, we have discovered the catalytic activemore » site and have identified Asp288 as the deprotonation residue and Lys290 as the autoacetylation residue. We have further proposed the unique H3-K56 anchoring pocket and the potential H3?N binding groove. Our work has provided structural insights to understand the acetylation mechanism of H3-K56 by Rtt109.« less
  • Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.
  • C-terminal binding protein (CtBP) has been shown to bind to a highly conserved five-amino-acid motif (PXDLS) located very close to the C-terminus of adenovirus early region 1A proteins. It has also been demonstrated that amino acids C-terminal and N-terminal to this original proposed binding site contribute to the interaction. However, conflicting evidence has been presented to show that acetylation of an adjacent lysine residue in Ad5E1A may or may not influence binding. It has now been demonstrated here that acetylation of a lysine, equivalent to position 261 in Ad12 E1A and position 285 in Ad5E1A, in a synthetic peptide disruptsmore » the binding to CtBP1 and CtBP2 and alters the K {sub i} of the peptide, indicative of a reduction in the affinity of the peptide for CtBP1 and CtBP2, but only to a rather limited extent (less than 2-fold). The solution structures of synthetic peptides equivalent to wild-type and acetylated forms of the Ad12 E1A peptide have been determined by proton NMR spectroscopy. The wild-type form of the peptide adopts a series of {beta}-turns over the region Val{sup 254}-Arg{sup 262}. Within the acetylated isoform, the {beta}-turn conformation is less extensive, Val{sup 26}-Arg{sup 262} adopting a random confirmation. We conclude that secondary structure ({beta}-turns) and an appropriate series of amino acid side chains over an extended binding site (PXDLSXK) are necessary for recognition by CtBP, acetylation of lysine interfering with both of these features, but not to such an extent as to totally inhibit interaction. Moreover, it is possible that the {beta}-turn conformation at the C-terminus of AdE1A contributes to binding to {alpha} importin and nuclear import. Acetylation of lysine {sup 261} could disrupt interaction through structural destabilization as well as charge neutralization and subsequent nuclear localization.« less