Schilling - Ecoli_Glucose_Acetylome

Protein acetylation in response to carbon overflow: detecting dynamic acetylation changes by mass spectrometry

Birgit Schilling, David Christensen, Bradford W. Gibson, Alan J. Wolfe et al.

Experimental Design for the E. coli glucose dependent acetylome project: 

To identify specific lysines with significant acetylation changes, we grew cells in TB7 with and without glucose, applied label-free, quantitative mass spectrometry (MS1 Filtering), monitored acetylated peptides, and compared acetylation changes (i) over time during growth in the presence of glucose and (ii) between glucose and no glucose growth conditions. This approach revealed increases over time in both the number of detected lysine acetylation sites and the dynamic relative increase of determined acetylation levels. During the time course, the change in acetylation varied greatly between individual lysines from the same proteins, consistent with differences in specificity. Glucose-regulated lysine acetylation sites were particularly predominant in central metabolic pathways and overlapped extensively with acetylPhosphgate-regulated lysine acetylation sites.

MS/MS spectral library for all identified E. coli 2813 lysine acetylation sites from 780 acetylated proteins (see above imported as Targeted MS Runs)

go to Panorama spectral library links directly:  MS/MS part1 and MS/MS additional sites

Quantitative details and chromatogram views for acetylated peptides from >1000 acetylation sites are provided to be viewed in Panorama: File A and File B   

Quantification of glucose regulated acetylation – dynamic acetylation changes following a glucose time course displayed for >400 proteins (download graphical displays).

Legend: Differential rates of acetylation: Individual Kac sites from the same protein show different rates of increasing acetylation during the glucose time course experiments (all Kac fold changes were normalized for potential protein level changes). Dynamic change of acetylation is displayed for >400 E coli proteins with the following Kac fold changes monitored between the following Glc-supplemented time points, 2h/2h (fold change set to 1), 5h/2h, 8h/2h, and 12h/2h.

MS/MS identification details for lysine acetylated peptides from database search results, Protein Pilot and Mascot (download identification excel file)

3 dimensional protein structures (Pymol) of selected acetylated proteins from the TCA cycle, Glycolysis and Phosphate Pentose Pathway indicating location of acetylation (Kac) sites (download pdf file)

Protein acetylation in response to carbon overflow: detecting dynamic acetylation changes by mass spectrometry

  • Organism: Escherichia coli
  • Instrument: TripleTOF 6600,TripleTOF 5600
  • SpikeIn: No
Abstract
Non-enzymatic acetyl phosphate (acP)-dependent Nε-lysine acetylation predominates in the bacterium Escherichia coli. To describe this post-translational modification, we grew wild-type cells in buffered tryptone broth (TB7) plus glucose or lactate, and monitored acetylation over time by Western immunoblot and mass spectrometry. Although acetylation began during exponential growth, most occurred in stationary phase which paralleled glucose consumption and acetate excretion that began upon entry into stationary phase. This explains how acP-dependent acetylation can occur throughout stationary phase. The large increase in acetylation during stationary phase occurred only if glucose or lactate were added during exponential growth. Transcription of the small RNA rprA, which regulates the alternative stationary phase sigma factor, exhibited similar behavior. To identify specific lysines with significant acetylation changes, we grew cells in TB7 with and without glucose, applied label-free, quantitative mass spectrometry (MS1 Filtering) to monitor changes in acetylated peptide levels over time during growth in the presence of glucose and without glucose. This approach revealed increases over time in both the number of detected lysine acetylation sites and a dynamic increase in their acetylation levels. During the time course, the change in acetylation varied greatly between individual lysines from the same proteins, consistent with differences in specificity. Since glucose-regulated lysine acetylation sites were particularly predominant in central metabolic pathways and overlapped extensively with acP-regulated lysine acetylation sites, we deleted crp, which encodes the major carbon regulator, and observed a dramatic loss of acetylation that was restored by deleting ackA, which increases acP. We propose that acP-dependent acetylation is a response to carbon flux that has the potential to regulate central metabolism.
Experiment Description
Experimental Design: To identify specific lysines with significant acetylation changes, we grew cells in TB7 with and without glucose, applied label-free, quantitative mass spectrometry (MS1 Filtering), monitored acetylated peptides, and compared acetylation changes (i) over time during growth in the presence of glucose and (ii) between glucose and no glucose growth conditions. This approach revealed increases over time in both the number of detected lysine acetylation sites and the dynamic relative increase of determined acetylation levels. During the time course, the change in acetylation varied greatly between individual lysines from the same proteins, consistent with differences in specificity. Glucose-regulated lysine acetylation sites were particularly predominant in central metabolic pathways and overlapped extensively with acetylPhosphgate-regulated lysine acetylation sites.
Sample Description
Lysine acetyl antibody enriched peptide fractions from E. coli grown under different growth conditions: WT E. coli K-12 grown in buffered tryptone broth (TB7) supplemented with 0.4% glucose (Glc) or in TB7 media without Glc. Conditions WT 2h Glc, 5h Glc, 8h Glc, 12h Glc, and 12h no Glc 3 biological replicates 3 technical MS replicates
Created on 3/9/15, 12:25 PM
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
Ecoli_glucose_1_final_acetylome_v14_Panorama_part1.sky.zip (21 MB)2015-03-097213,5664,51613,5480
Ecoli_glucose_2_acetylome_extra for Panorama_add_on_part2.sky.zip (968 KB)2015-03-091591861965880
DC3_acetyl_new_DC2based_0902_2014_v14_for_PanoramaXICb_2015-03-05_12-16-42.sky.zip (888 MB)2015-03-094811,2651,2683,80415
DC2_acetyl_0704_2014_v13_for_PanoramaXICb_2015-03-05_12-18-33.sky.zip (860 MB)2015-03-095721,6621,6664,99815