Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)
Abstract
Post-translational modification of lysine residues by Nε-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride.more »
- Authors:
-
- Buck Institute for Research on Aging, 94945, Novato, CA, USA
- Amgen, 94080, South San Francisco, CA, USA
- Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, 60153, Maywood, IL, USA
- Buck Institute for Research on Aging, 94945, Novato, CA, USA, Department of Pharmaceutical Chemistry, University of California, 94143, San Francisco, CA, USA
- Publication Date:
- Research Org.:
- Buck Inst. for Research on Aging, Novato, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Inst. of Health (NIH) (United States)
- OSTI Identifier:
- 1315863
- Alternate Identifier(s):
- OSTI ID: 1425373
- Grant/Contract Number:
- SC0012443; R24DK085610; T32G000266; 1S10 OD016281
- Resource Type:
- Published Article
- Journal Name:
- Journal of the American Society for Mass Spectrometry
- Additional Journal Information:
- Journal Name: Journal of the American Society for Mass Spectrometry Journal Volume: 27 Journal Issue: 11; Journal ID: ISSN 1044-0305
- Publisher:
- American Chemical Society
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; stoichiometry; acetylation; succinylation; SWATH; data-independent acquisition; mass spectrometry; skyline
Citation Formats
Meyer, Jesse G., D’Souza, Alexandria K., Sorensen, Dylan J., Rardin, Matthew J., Wolfe, Alan J., Gibson, Bradford W., and Schilling, Birgit. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH). United States: N. p., 2016.
Web. doi:10.1007/s13361-016-1476-z.
Meyer, Jesse G., D’Souza, Alexandria K., Sorensen, Dylan J., Rardin, Matthew J., Wolfe, Alan J., Gibson, Bradford W., & Schilling, Birgit. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH). United States. https://doi.org/10.1007/s13361-016-1476-z
Meyer, Jesse G., D’Souza, Alexandria K., Sorensen, Dylan J., Rardin, Matthew J., Wolfe, Alan J., Gibson, Bradford W., and Schilling, Birgit. Fri .
"Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)". United States. https://doi.org/10.1007/s13361-016-1476-z.
@article{osti_1315863,
title = {Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)},
author = {Meyer, Jesse G. and D’Souza, Alexandria K. and Sorensen, Dylan J. and Rardin, Matthew J. and Wolfe, Alan J. and Gibson, Bradford W. and Schilling, Birgit},
abstractNote = {Post-translational modification of lysine residues by Nε-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.},
doi = {10.1007/s13361-016-1476-z},
journal = {Journal of the American Society for Mass Spectrometry},
number = 11,
volume = 27,
place = {United States},
year = {Fri Sep 02 00:00:00 EDT 2016},
month = {Fri Sep 02 00:00:00 EDT 2016}
}
https://doi.org/10.1007/s13361-016-1476-z
Web of Science
Works referenced in this record:
Acetylation dynamics and stoichiometry in S accharomyces cerevisiae
journal, January 2014
- Weinert, Brian T.; Iesmantavicius, Vytautas; Moustafa, Tarek
- Molecular Systems Biology, Vol. 10, Issue 1
SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation
journal, March 2010
- Hirschey, Matthew D.; Shimazu, Tadahiro; Goetzman, Eric
- Nature, Vol. 464, Issue 7285
Controlling Deuterium Isotope Effects in Comparative Proteomics
journal, August 2002
- Zhang, Roujian; Sioma, Cathy S.; Thompson, Robert A.
- Analytical Chemistry, Vol. 74, Issue 15
The First Identification of Lysine Malonylation Substrates and Its Regulatory Enzyme
journal, September 2011
- Peng, Chao; Lu, Zhike; Xie, Zhongyu
- Molecular & Cellular Proteomics, Vol. 10, Issue 12
Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline: APPLICATION TO PROTEIN ACETYLATION AND PHOSPHORYLATION
journal, March 2012
- Schilling, Birgit; Rardin, Matthew J.; MacLean, Brendan X.
- Molecular & Cellular Proteomics, Vol. 11, Issue 5
Lysine Acetylation Is a Highly Abundant and Evolutionarily Conserved Modification in Escherichia Coli
journal, August 2008
- Zhang, Junmei; Sprung, Robert; Pei, Jimin
- Molecular & Cellular Proteomics, Vol. 8, Issue 2
Calorie restriction alters mitochondrial protein acetylation
journal, October 2009
- Schwer, Bjoern; Eckersdorff, Mark; Li, Yu
- Aging Cell, Vol. 8, Issue 5
Skyline: an open source document editor for creating and analyzing targeted proteomics experiments
journal, February 2010
- MacLean, Brendan; Tomazela, Daniela M.; Shulman, Nicholas
- Bioinformatics, Vol. 26, Issue 7
The growing landscape of lysine acetylation links metabolism and cell signalling
journal, July 2014
- Choudhary, Chunaram; Weinert, Brian T.; Nishida, Yuya
- Nature Reviews Molecular Cell Biology, Vol. 15, Issue 8
Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution
journal, October 2007
- Keshishian, Hasmik; Addona, Terri; Burgess, Michael
- Molecular & Cellular Proteomics, Vol. 6, Issue 12
Probability-based protein identification by searching sequence databases using mass spectrometry data
journal, December 1999
- Perkins, David N.; Pappin, Darryl J. C.; Creasy, David M.
- Electrophoresis, Vol. 20, Issue 18
Lysine Acetylation: Codified Crosstalk with Other Posttranslational Modifications
journal, August 2008
- Yang, Xiang-Jiao; Seto, Edward
- Molecular Cell, Vol. 31, Issue 4
Quantitative Phosphoproteomics Reveals Widespread Full Phosphorylation Site Occupancy During Mitosis
journal, January 2010
- Olsen, J. V.; Vermeulen, M.; Santamaria, A.
- Science Signaling, Vol. 3, Issue 104
iProphet: Multi-level Integrative Analysis of Shotgun Proteomic Data Improves Peptide and Protein Identification Rates and Error Estimates
journal, August 2011
- Shteynberg, David; Deutsch, Eric W.; Lam, Henry
- Molecular & Cellular Proteomics, Vol. 10, Issue 12
Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome
journal, June 2014
- Baeza, Josue; Dowell, James A.; Smallegan, Michael J.
- Journal of Biological Chemistry, Vol. 289, Issue 31
MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments
journal, May 2015
- Rardin, Matthew J.; Schilling, Birgit; Cheng, Lin-Yang
- Molecular & Cellular Proteomics, Vol. 14, Issue 9
Identification of Lysine Succinylation Substrates and the Succinylation Regulatory Enzyme CobB in Escherichia coli
journal, October 2013
- Colak, Gozde; Xie, Zhongyu; Zhu, Anita Y.
- Molecular & Cellular Proteomics, Vol. 12, Issue 12
Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions
journal, July 2009
- Choudhary, C.; Kumar, C.; Gnad, F.
- Science, Vol. 325, Issue 5942
Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase
journal, July 2012
- Lima, Bruno P.; Thanh Huyen, Tran Thi; Bäsell, Katrin
- Journal of Biological Chemistry, Vol. 287, Issue 38
Involvement of protein acetylation in glucose-induced transcription of a stress-responsive promoter: Acetylation of RNA polymerase
journal, July 2011
- Lima, Bruno P.; Antelmann, Haike; Gronau, Katrin
- Molecular Microbiology, Vol. 81, Issue 5
The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide–dependent deacetylase
journal, August 2002
- Schwer, Björn; North, Brian J.; Frye, Roy A.
- The Journal of Cell Biology, Vol. 158, Issue 4
A cross-platform toolkit for mass spectrometry and proteomics
journal, October 2012
- Chambers, Matthew C.; Maclean, Brendan; Burke, Robert
- Nature Biotechnology, Vol. 30, Issue 10
Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways
journal, April 2013
- Rardin, M. J.; Newman, J. C.; Held, J. M.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 16
SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways
journal, June 2013
- Park, Jeongsoon; Chen, Yue; Tishkoff, Daniel X.
- Molecular Cell, Vol. 50, Issue 6
Empirical Statistical Model To Estimate the Accuracy of Peptide Identifications Made by MS/MS and Database Search
journal, October 2002
- Keller, Andrew; Nesvizhskii, Alexey I.; Kolker, Eugene
- Analytical Chemistry, Vol. 74, Issue 20
Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows
journal, September 2015
- Schilling, Birgit; MacLean, Brendan; Held, Jason M.
- Analytical Chemistry, Vol. 87, Issue 20
SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks
journal, December 2013
- Rardin, Matthew J.; He, Wenjuan; Nishida, Yuya
- Cell Metabolism, Vol. 18, Issue 6
Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli
journal, July 2013
- Weinert, Brian T.; Iesmantavicius, Vytautas; Wagner, Sebastian A.
- Molecular Cell, Vol. 51, Issue 2
DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics
journal, January 2015
- Tsou, Chih-Chiang; Avtonomov, Dmitry; Larsen, Brett
- Nature Methods, Vol. 12, Issue 3
Lysine Succinylation Is a Frequently Occurring Modification in Prokaryotes and Eukaryotes and Extensively Overlaps with Acetylation
journal, August 2013
- Weinert, Brian T.; Schölz, Christian; Wagner, Sebastian A.
- Cell Reports, Vol. 4, Issue 4
Histone deacetylases and cancer
journal, August 2007
- Glozak, M. A.; Seto, E.
- Oncogene, Vol. 26, Issue 37
50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond
journal, December 2014
- Verdin, Eric; Ott, Melanie
- Nature Reviews Molecular Cell Biology, Vol. 16, Issue 4
Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter
journal, July 2013
- Hu, L. I.; Chi, B. K.; Kuhn, M. L.
- Journal of Bacteriology, Vol. 195, Issue 18
SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target
journal, July 2015
- Nishida, Yuya; Rardin, Matthew J.; Carrico, Chris
- Molecular Cell, Vol. 59, Issue 2
Deep, Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow
journal, May 2015
- Svinkina, Tanya; Gu, Hongbo; Silva, Jeffrey C.
- Molecular & Cellular Proteomics, Vol. 14, Issue 9
Regulation of chromatin by histone modifications
journal, February 2011
- Bannister, Andrew J.; Kouzarides, Tony
- Cell Research, Vol. 21, Issue 3
Site-Specific Identification of Lysine Acetylation Stoichiometries in Mammalian Cells
journal, February 2016
- Zhou, Tong; Chung, Ying-hua; Chen, Jianji
- Journal of Proteome Research, Vol. 15, Issue 3
Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation
journal, April 2014
- Kuhn, Misty L.; Zemaitaitis, Bozena; Hu, Linda I.
- PLoS ONE, Vol. 9, Issue 4
Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli
journal, November 2014
- Castaño‐Cerezo, Sara; Bernal, Vicente; Post, Harm
- Molecular Systems Biology, Vol. 10, Issue 11
Analysis of acetylation stoichiometry suggests that SIRT 3 repairs nonenzymatic acetylation lesions
journal, September 2015
- Weinert, Brian T.; Moustafa, Tarek; Iesmantavicius, Vytautas
- The EMBO Journal, Vol. 34, Issue 21
Widespread and Enzyme-independent N ϵ -Acetylation and N ϵ -Succinylation of Proteins in the Chemical Conditions of the Mitochondrial Matrix
journal, August 2013
- Wagner, Gregory R.; Payne, R. Mark
- Journal of Biological Chemistry, Vol. 288, Issue 40
Substrate and Functional Diversity of Lysine Acetylation Revealed by a Proteomics Survey
journal, August 2006
- Kim, Sung Chan; Sprung, Robert; Chen, Yue
- Molecular Cell, Vol. 23, Issue 4
Protein acetylation dynamics in response to carbon overflow in E scherichia coli : Acetylation dynamics and overflow metabolism
journal, September 2015
- Schilling, Birgit; Christensen, David; Davis, Robert
- Molecular Microbiology, Vol. 98, Issue 5
The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites
journal, November 2014
- AbouElfetouh, Alaa; Kuhn, Misty L.; Hu, Linda I.
- MicrobiologyOpen, Vol. 4, Issue 1
Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells
journal, April 2011
- Wang, Yuexi; Yang, Feng; Gritsenko, Marina A.
- PROTEOMICS, Vol. 11, Issue 10
MS-GF+ makes progress towards a universal database search tool for proteomics
journal, October 2014
- Kim, Sangtae; Pevzner, Pavel A.
- Nature Communications, Vol. 5, Issue 1
Mitochondrial sirtuins: regulators of protein acylation and metabolism
journal, September 2012
- He, Wenjuan; Newman, John C.; Wang, Margaret Z.
- Trends in Endocrinology & Metabolism, Vol. 23, Issue 9
A Method to Determine Lysine Acetylation Stoichiometries
journal, January 2014
- Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.
- International Journal of Proteomics, Vol. 2014
Regulation of acetate metabolism in Escherichia coli BL21 by protein Nε-lysine acetylation
journal, December 2014
- Castaño-Cerezo, Sara; Bernal, Vicente; Röhrig, Teresa
- Applied Microbiology and Biotechnology, Vol. 99, Issue 8