title: Accurate Quantification of Site-specific Acetylation Stoichiometry Reveals the Impact of Sirtuin Deacetylase CobB on theE. coliAcetylome.
abstract: Lysine acetylation is a protein posttranslational modification (PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometry on a proteome-wide scale remains challenging. Most methods employ a comparison of chemically acetylated peptides to native acetylated peptides, however, the potentially large differences in abundance between these peptides presents a challenge for accurate quantification. Stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry (MS) is one of the most widely used quantitative proteomic methods. Here we show that serial dilution of SILAC-labeled peptides (SD-SILAC) can be used to identify accurately quantified peptides and to estimate the quantification error rate. We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild-type and Sirtuin deacetylase CobB-deficient cells. To further analyze CobB-regulated sites under conditions of globally increased or decreased acetylation, we measured stoichiometry in phophotransacetylase (ptaΔ) and acetate kinase (ackAΔ) mutant strains in the presence and absence of the Sirtuin inhibitor nicotinamide. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurred at a low stoichiometry. Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such as stationary-phase arrest and deletion ofackA, resulted in globally increased acetylation stoichiometry. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. Similar to Sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry in WT,ptaΔ, andackAΔ cells. Together, our results provide a detailed view of acetylation stoichiometry inE. coliand suggest an evolutionarily conserved function of Sirtuin deacetylases in suppressing low stoichiometry acetylation.
authors.affiliation: From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark chuna.choudhary@cpr.ku.dk briantate.weinert@cpr.ku.dk.
authors.forename: Brian Tate
authors.initials: BT
authors.lastname: Weinert
authors.affiliation: From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
authors.forename: Shankha
authors.initials: S
authors.lastname: Satpathy
authors.affiliation: From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
authors.forename: Bogi Karbech
authors.initials: BK
authors.lastname: Hansen
authors.affiliation: From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
authors.forename: David
authors.initials: D
authors.lastname: Lyon
authors.affiliation: From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
authors.forename: Lars Juhl
authors.initials: LJ
authors.lastname: Jensen
authors.affiliation: From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark chuna.choudhary@cpr.ku.dk briantate.weinert@cpr.ku.dk.
authors.forename: Chunaram
authors.initials: C
authors.lastname: Choudhary
mesh_terms: D000107:Acetylation; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007553:Isotope Labeling; D020543:Proteome / Q000378:metabolism*; D037761:Sirtuins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome; D037761:Sirtuins; C483998:cobB protein, E Coli