Acetylation is a dynamic modification in B. subtilis. Acetylation is a dynamic modification in B. subtilis. (A) Wild-type cells (BD630) were grown to the log or stat phase in minimal glucose medium. Arrows on the growth curve indicate the mid-log-phase and early-stat-phase time points used for all studies. Cells were harvested and rapidly frozen for cryogenic cell lysis. This technique was used to obtain a more consistent disruption of the cells, which leads to improved efficiency of protein extraction and reproducibility of acetyl-peptide capture. The image depicts a sample after grinding showing nearly complete cell lysis (>85%). Lysed cell material was resuspended in a heated SDS buffer, and proteins were precipitated and subjected to digestion with trypsin. An aliquot of peptides was saved for whole-cell protein analysis, while the remaining peptides were incubated with agarose beads coated with a mixture of commercially available anti-acetyllysine antibodies. Acetyllysine peptide elutions and peptides from the whole-cell digests were analyzed by label-free quantitative mass spectrometry using nanoscale liquid chromatography (nLC) coupled directly to an LTQ Orbitrap Velos ETD mass spectrometer. Peptide MS/MS sequencing was performed for both sample sets. (B) Wild-type cells were grown to the log and stat phases and lysates prepared as described in Materials and Methods. Equal amounts of protein were loaded in duplicate, with one set probed with a mixture of anti-acetyllysine (acetyl-K) antibodies and the other probed with anti-elongation factor G (anti-EFG) as a loading control. Log- and stat-phase samples were analyzed on the same membrane. (C) Venn diagrams comparing the number of identified acetylated sites (left) and proteins (right) in the log (red) and stat (blue) phases. Valerie J. Carabetta et al. mSystems 2016; doi:10.1128/mSystems.00005-16