Format

Send to

Choose Destination
Mol Cell Proteomics. 2017 May;16(5):759-769. doi: 10.1074/mcp.M117.067587. Epub 2017 Mar 2.

Accurate Quantification of Site-specific Acetylation Stoichiometry Reveals the Impact of Sirtuin Deacetylase CobB on the E. coli Acetylome.

Author information

1
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.
2
From the ‡The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.

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 of ackA, 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, and ackAΔ cells. Together, our results provide a detailed view of acetylation stoichiometry in E. coli and suggest an evolutionarily conserved function of Sirtuin deacetylases in suppressing low stoichiometry acetylation.

PMID:
28254776
PMCID:
PMC5417819
DOI:
10.1074/mcp.M117.067587
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center