All Title Author
Keywords Abstract

A Method to Determine Lysine Acetylation Stoichiometries

DOI: 10.1155/2014/730725

Full-Text   Cite this paper   Add to My Lib


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. 1. Introduction Lysine acetylation (KAc) of proteins is a ubiquitous posttranslational modification (PTM) that controls many cellular processes. The dynamic regulation of KAc by lysine acetyltransferases (KATs) and deacetylases (KDACs) modulates many important cellular functions, such as cell metabolism and gene expression [1, 2]. Recent advances in mass spectrometry combined with immunoaffinity purification are enabling the identification and relative quantification of thousands of acetylation sites in a single experiment [3–5]. These new data have boosted the discovery of regulatory functions of KAc for many proteins, including a variety of metabolic enzymes [2]. Although significant progress has been made, a major remaining hurdle in the field is the determination of acetylation stoichiometry on proteins. The knowledge of KAc stoichiometry is considered essential to better understand the mechanism and impact of this modification on the control protein functions, such as enzymatic activity [2, 6]. Indeed, this problem is not exclusive to KAc as there are almost no systematic determinations of the stoichiometry of PTMs. This has remained a challenge because methods to determine stoichiometry of PTMs are not compatible with enrichment procedures, since both modified and unmodified versions of polypeptides need to be present in the sample. Global studies have been successfully performed to determine the stoichiometries


[1]  X. J. Yang and E. Seto, “Lysine acetylation: codified crosstalk with other posttranslational modifications,” Molecular Cell, vol. 31, pp. 449–461, 2008.
[2]  Y. Xiong and K. L. Guan, “Mechanistic insights into the regulation of metabolic enzymes by acetylation,” Journal of Cell Biology, vol. 198, pp. 155–164, 2012.
[3]  S. C. Kim, R. Sprung, Y. Chen, et al., “Substrate and functional diversity of lysine acetylation revealed by a proteomics survey,” Molecular Cell, vol. 23, no. 4, pp. 607–618, 2006.
[4]  C. Choudhary, C. Kumar, F. Gnad, et al., “Lysine acetylation targets protein complexes and co-regulates major cellular functions,” Science, vol. 325, no. 5942, pp. 834–840, 2009.
[5]  A. Lundby, K. Lage, B. T. Weinert, et al., “Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns,” Cell Reports, vol. 2, pp. 419–431, 2012.
[6]  M. J. Rardin, J. C. Newman, J. M. Held, et al., “Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways,” Proceedings of the National Academy of Sciences of the United States of America, vol. 110, pp. 6601–6606, 2013.
[7]  R. Wu, W. Haas, N. Dephoure, et al., “A large-scale method to measure absolute protein phosphorylation stoichiometries,” Nature Methods, vol. 8, pp. 677–683, 2011.
[8]  J. V. Olsen, M. Vermeulen, A. Santamaria, et al., “Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis,” Science Signaling, vol. 3, no. 104, article ra3, 2010.
[9]  B. T. Weinert, V. Iesmantavicius, T. Moustafa, et al., “Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae,” Molecular Systems Biology, vol. 10, p. 716, 2014.
[10]  J. Y. Kim, K. W. Kim, H. J. Kwon, et al., “Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation,” Analytical Chemistry, vol. 74, no. 21, pp. 5443–5449, 2002.
[11]  K. Zhang, H. Tang, L. Huang, et al., “Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry,” Analytical Biochemistry, vol. 306, no. 2, pp. 259–269, 2002.
[12]  M. B. Trelle and O. N. Jensen, “Utility of immonium ions for assignment of epsilon-N-acetyllysine-containing peptides by tandem mass spectrometry,” Analytical Chemistry, vol. 80, pp. 3422–3430, 2008.
[13]  D. Shechter, H. L. Dormann, C. D. Allis, et al., “Extraction, purification and analysis of histones,” Nature Protocols, vol. 2, pp. 1445–1457, 2007.
[14]  C. Ansong, H. Yoon, S. Porwollik, et al., “Global systems-level analysis of Hfq and SmpB deletion mutants in Salmonella: implications for virulence and global protein translation,” PLoS ONE, vol. 4, Article ID e4809, 2009.
[15]  Y. Wang, F. Yang, M. A. Gritsenko, et al., “Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells,” Proteomics, vol. 11, no. 10, pp. 2019–2026, 2011.
[16]  A. M. Mayampurath, N. Jaitly, S. O. Purvine et al., “DeconMSn: a software tool for accurate parent ion monoisotopic mass determination for tandem mass spectra,” Bioinformatics, vol. 24, no. 7, pp. 1021–1023, 2008.
[17]  S. Kim, N. Mischerikow, N. Bandeira, et al., “The generating function of CID, ETD, and CID/ETD pairs of tandem mass spectra: applications to database search,” Molecular & Cellular Proteomics, vol. 9, pp. 2840–2852, 2010.
[18]  S. Kim, N. Gupta, and P. A. Pevzner, “Spectral probabilities and generating functions of tandem mass spectra: a strike against decoy databases,” Journal of Proteome Research, vol. 7, pp. 3354–3363, 2008.
[19]  M. E. Monroe, J. L. Shaw, D. S. Daly, J. N. Adkins, and R. D. Smith, “MASIC: a software program for fast quantitation and flexible visualization of chromatographic profiles from detected LC-MS(/MS) features,” Computational Biology and Chemistry, vol. 32, no. 3, pp. 215–217, 2008.
[20]  A. Michalski, N. Neuhauser, J. Cox, et al., “A systematic investigation into the nature of tryptic HCD spectra,” Journal of Proteome Research, vol. 11, no. 11, pp. 5479–5491, 2012.
[21]  I. A. Papayannopoulos, “The interpretation of collision-induced dissociation tandem mass-spectra of peptides,” Mass Spectrometry Reviews, vol. 14, pp. 49–73, 1995.
[22]  V. Faca, M. Coram, D. Phanstiel, et al., “Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS,” Journal of Proteome Research, vol. 5, pp. 2009–2018, 2006.
[23]  Q. Zhang, W. J. Qian, T. V. Knyushko, et al., “A method for selective enrichment and analysis of nitrotyrosine-containing peptides in complex proteome samples,” Journal of Proteome Research, vol. 6, pp. 2257–2268, 2007.
[24]  S. Y. Ow, M. Salim, J. Noirel, et al., “iTRAQ underestimation in simple and complex mixtures: the good, the bad and the ugly,” Journal of Proteome Research, vol. 8, pp. 5347–5355, 2009.
[25]  C. M. Smith, P. R. Gafken, and Z. Zhang, “Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4,” Analytical Biochemistry, vol. 316, pp. 23–33, 2003.
[26]  G. E. Zentner and S. Henikoff, “Regulation of nucleosome dynamics by histone modifications,” Nature Structural & Molecular Biology, vol. 20, pp. 259–266, 2013.
[27]  Y. S. Gao, C. C. Hubbert, J. Lu, et al., “Histone deacetylase 6 regulates growth factor-induced actin remodeling and endocytosis,” Molecular and Cellular Biology, vol. 27, no. 24, pp. 8637–8647, 2007.
[28]  J. C. Newman, W. He, and E. Verdin, “Mitochondrial protein acylation and intermediary metabolism: regulation by sirtuins and implications for metabolic disease,” The Journal of Biological Chemistry, vol. 287, pp. 42436–42434, 2012.


comments powered by Disqus