Schilling - NGackA

Schilling - NGackA

Identification and characterization of protein acetylation in Neisseria gonorrhoeae

 

Deborah M. B. Post, Birgit Schilling, Lorri M. Reinders, Alexandria K. D’Souza, Margaret R. Ketterer, Steven J. Kiel,

Aroon T. Chande, Michael A. Apicella and Bradford W. Gibson

 

  • Three biological replicates of Neisseria gonorrhoeae 1291wt and 1291ackA strains were grown in broth
  • An anti-lysine acetylation antibody was used to enriched for acetylated peptides
  • Samples were analyzed on a TripleTOF 5600
  • Comparative analyses (MS1 filtering) of the acetylated peptides from 1291wt and 1291ackA were performed using Skyline

Clustergrammer Heatmap
 
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SigPath_TitrationCurve_IMAC_04282021_2021-06-11_11-37-39.sky.zip2021-09-21 20:44:231321863761,57215
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Calibration peptide TPLTATLSK.sky.zip2021-08-17 20:18:281121033
Calibration, peptide SAVQGPPER.sky.zip2021-08-17 20:18:281121254
Calibration peptide VVLEGGIDPILR.sky.zip2021-08-17 20:18:28112630
Calibration peptide LGHPDTLNQGEFK.sky.zip2021-08-17 20:18:28112824
Calibration peptide NQNTFLR.sky.zip2021-08-17 20:18:28112830
Calibration, peptide DPPQYPVVPVHLDR.sky.zip2021-08-17 20:18:281121026
Calibration peptide DLQNFLK.sky.zip2021-08-17 20:18:281121030
Calibration peptide DASGATFTWTPSSGK.sky.zip2021-08-17 20:18:281121027
Calibration peptide AVLTIDEK.sky.zip2021-08-17 20:18:281121430
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Figure 5.sky.zip2021-07-10 09:08:069203410230
Figure 4.sky.zip2021-07-10 09:08:069203410222
Figure 3D.sky.zip2021-07-10 09:08:059203410227
Figure 3C.sky.zip2021-07-10 09:08:059203410227
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Identification and characterization of protein acetylation in Neisseria gonorrhoeae
  • Organism: Neisseria gonorrhoeae
  • Instrument: Triple TOF 5600
  • SpikeIn: No
Abstract
Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work demonstrated that lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wt samples demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values<0.05 in at least 2/3 of the biological replicates and were designated as “regulated” or AckA-dependent. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a reduced growth rate under aerobic conditions, an inability to grow anaerobically and had a defect in biofilm maturation.
Experiment Description
To identify specific lysine acetylation sites that were acetyl-phosphate (Ac-P) dependent, an ackA mutant, that accumulates AcP, was generated. Label-free relative quantitation (MS1 filtering) was used to compare acetylated peptides from wt with ackA samples.
Sample Description
Triplicate biological replicates of Neisseria gonorrhoeae strains 1291wt and 1291ackA were grown in 500 ml cultures of GC-broth supplemented with 1% IsoVitaleX and Kellogg’s supplement at 37°C, rotating at 150 rpm, overnight. Peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Two mass spectrometric injection replicates were acquired for each sample.
Created on 6/14/16, 11:06 AM