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
Download 20:44:231321863761,57215 20:44:2375911827086 20:43:251602274581,90639 20:42:101562274641,88216 20:42:10729018270616 20:40:01688316664830 20:40:011492184481,80830 20:38:57688717467610 20:38:578111223088210 16:33:19131402166 18:47:003110310347155
Calibration peptide 20:18:281121033
Calibration, peptide 20:18:281121254
Calibration peptide 20:18:28112630
Calibration peptide 20:18:28112824
Calibration peptide 20:18:28112830
Calibration, peptide 20:18:281121026
Calibration peptide 20:18:281121030
Calibration peptide 20:18:281121027
Calibration peptide 20:18:281121430
Calibration peptide 20:18:281121024
Proteomic analysis of 134 newborn 20:18:289102093134
Trypsin digestion time 20:18:28910209321
Matrix effect on protein 20:18:28910209821
Initial screening_SRMatlas 12:40:4011313651
Initial screening_Skyline 12:40:4012424751
Quantitative performance of SIL-TCT and SIL-ExC5 dissolved in water and SDC 12:40:40110209612
SiL-Ex peptides solubility 12:40:40110209636
Incubation time optimization-additional measurement for SIL-ExC3N3 peptide 12:40:401121018
Optimization of incubation time for trypsin 12:40:39110209672
Comparison of all types of internal 12:40:39110209615 12:40:39110209636
SPE 12:40:3911020966 13:59:2490565620 13:59:2490888850 08:07:143030606018 06:10:36328371724 08:33:26325911884730 08:33:26325911884125 08:33:26377014054226 08:33:26325911884715
20180731_UPR_ARoos_H& 07:57:138214214228
Nrxn3 HA IP splice segment analysis2021-07-13 08:24:0947147011
Nrxn3 AS5 knock-out analysis2021-07-13 08:17:391153216631
Nrxn3 Exon 25 Titration Curve2021-07-13 08:15:0518169214
Figure 09:08:069203410230
Figure 09:08:069203410222
Figure 09:08:059203410227
Figure 09:08:059203410227
Figure 09:08:059203410222
Figure 09:08:059203410222 07:13:4711245
PRM Splice Assays2021-06-24 08:36:0739841659900
DDR2_response 08:35:5527469264434
210429to210501_ApoE_5x5_Precision.sky2021-06-22 12:52:48151024139 08:22:184510801 13:23:364510801
DDR2_response 11:52:4827469264434 09:20:413,86625,31129,238175,3950 13:46:483,59738,88950,764304,5840 15:46:21131402164 04:19:42902765792,8570 04:17:39741633411,6630 08:15:4419569997610 08:15:441944746756 20:58:381544344342,98414 20:58:381244234232,95933 20:58:381244234232,95933 20:58:381244234236,20233 20:58:381244234236,20233 20:58:381284604603,01420 20:21:56818116280285 11:14:24818116285096 07:04:33131402164
MRM 15:28:2822459026841
MRM 15:28:2822438625638
MRM 15:28:2822438625640 06:02:52112141 18:04:172738765261 18:03:1078161101 18:02:2011281 18:01:31112141 14:46:32404124891,40511 14:46:32224129686218 14:46:32626330991811 10:24:111371312
20190927 PIP extraction 06:45:2710114122
20190928 Donor 6-7 PIPs MRM 06:45:2610114130
20190929 Extraction 06:45:2610114120
20191002 Brain 06:45:2610114113
20191003 Donor 6-7 + calibration 06:45:2610114136
20191105 Calibration 06:45:2610114130
20191108 06:45:261011413
D2 15 06:45:2610114120
20191114 Extraction 06:45:2610114119
PIPs MRM 191001 Donor 06:45:2610114124
PIPs MRM 190930 06:45:2610114111 20:17:4890961921,20296 20:17:4890961921,12295 20:17:4890961921,05495
Identification and characterization of protein acetylation in Neisseria gonorrhoeae
  • Organism: Neisseria gonorrhoeae
  • Instrument: Triple TOF 5600
  • SpikeIn: No
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