Blackburn Lab - PknG physiological substrates

Blackburn Lab - PknG physiological substrates
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates 15:20:4490961922,76490 22:10:23131402165 21:25:59264762373 21:25:5911921636 21:25:582,77924,31330,00390,00933
Sky0 ACE2 TMPRSS2 cell 13:38:456252919732 16:24:3290961922,76493 15:34:1311261 15:34:13112668 15:34:1311262 15:34:1311268 15:34:13112663 15:34:13112672 15:34:131121069 15:34:13112101 15:34:13112102 15:34:13112460 15:34:13112412 15:34:13112669 15:34:1311262
Sky1 Synthetic SARS-CoV2 proteome 13:21:56121431351,2121
Sky3 52 patients nano PRM-MS2 13:20:236345734055
Sky4 5 positive patients nano PRM-MS2 13:20:22634492945
Sky6 11 positive patients micro PRM-MS2 13:11:206345527311
Sky5 91 patients micro PRM-MS2 13:11:206345527391
Sky2 Dilution series SARS Cov2 nano and 13:04:536345737118 20:16:492828562164 20:16:492627542064 20:16:492424481824 05:16:2773154154616102
Intra-day 05:14:01731541546166
Inter-day 05:13:11731541546165
Instrumental set-up 05:11:30731541546165 01:26:17217715461713 01:17:00217815662512 20:17:53751481622,58918 20:17:272141254 20:17:272141263 20:17:272151639 20:17:272151631 20:17:272141235 20:17:272141238 20:17:272141265 20:17:272141211 20:17:262141264 20:17:262141213 20:17:262041284
Batch 20:17:262141265 20:17:262141219 20:16:45417201228 20:16:44299668 20:16:10299668 15:59:1690961921,02896 19:31:421,6914,7464,87926,1230 17:38:291,5075,0745,10425,3220 10:58:1317494921860 05:51:586253,2053,20513,12660 07:21:051,2225,9545,95422,91166 16:35:09112434 16:35:09112644 16:35:09112638 16:35:09112428 16:35:09112644 16:35:09112632 16:35:09112643 16:35:09112442 16:35:09112442 16:35:09112436 16:35:09112433 16:35:09112442 16:35:09112643 16:35:09112444 16:35:09112444 16:35:09112441 16:23:121616328224 16:23:121616328423 16:23:121616328229 16:23:121616328027 16:23:121616328425 16:23:121616328632 16:23:121616328225 16:23:121616328223 16:23:121616328218 16:23:121616328244 16:23:121616328230 16:23:121616328415 16:23:121616328021 16:23:121616328430 16:23:121616328424 16:23:121616328227 16:23:121616328028 16:23:121616328229 16:23:121616328224 16:23:121616328425 16:23:121616328422 16:23:111616328428 16:23:111616328428 16:23:111616328224 16:23:111616328235 16:23:111616328422
PknG physiological substrates

  • Organism: Mycobacterium bovis BCG
  • Instrument: QExactive
  • SpikeIn: No
Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. The challenge now lies in linking kinases to the physiological substrates they phosphorylate in vivo, thereby elucidating their exact functions. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BGC versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO2 phosphopeptide enrichment techniques combined with label free quantitative phosphoproteomics work flow on LC/MS/MS. The comprehensive analysis of label free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. 55 phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRM’s). The kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified p-sites presented docking scores close to that seen in previously described PknG substrates, GarA and ribosomal protein L13. Six out of the 23 phosphoproteins had higher docking scores than GarA, suggesting that the proteins identified here are truly PknG substrates. Based on protein functional analysis of the PknG substrates identified, the study confirms that PknG play an important regulation role in mycobacterial metabolism, but also indicated its association with the machinery of protein translation and folding.
Experiment Description
Identified PknG substrates were validated by targeted mass spectrometry. Briefly, the discovery phosphoproteomic data was used to identify peptides with confidently localised phosphosites (phospho probability of >0.75) that were present in the M.bovis BCG Wt and absent in the PknG knock-out mutant. Fourteen of these peptides, derived from 7 proteins, were selected to validate the phosphoproteomic data. A spectral library was generated using the discovery phosphoproteomic data in Skyline (version, with the best representative spectrum for each identified peptide and phosphopeptide. Retention times were calculated based on the average retention time observed in the discovery phosphoproteomic analysis. An isolation list was generated with a 10-minute retention time window around each peptide’s calculated retention time. This isolation list was used to carry out a 2-plex scheduled PRM analysis with 100 ms injection time and a total cycle time of 2 seconds on a QExactive hybrid Orbitrap mass spectrometer (Thermo). The AGC target was set to maximum, and a 2 m/z mass error window was allowed. Targeted MS2 data was acquired at a resolution of 35000. The chromatography setup was identical to that of the discovery phosphoproteomic analysis. The resulting PRM data was analysed in Skyline with the background M. bovis BCG database obtained from Uniprot ( The spectral library was used to confirm the identity of the targeted peptides.
Sample Description
M. bovis BCG reference strain (Pasteur 1172) and M.bovis BCG PKnG knock-out mutant strain generously donated by Prof Jean Pieters (Walburger et al., 2004) were grown n 7H9 DifcoTM Middlebrook liquid media (Becton Dickinson; BD), supplemented with OADC and Tween 80 to prevent clumping at 37 °C while shaking. Growth was monitored daily by measuring optical Density (OD600) and the growth curve plotted. Cells were harvested at mid-log (OD~ 0.6) by centrifugation for 10 min (4000 g) and washed twice in phosphate buffered saline (PBS) pH 7.4 (Sigma).
Created on 3/15/18, 9:40 AM