Blackburn Lab - PknG physiological substrates

Blackburn Lab - PknG physiological substrates
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LINCS_P100_DIA_Plate97_annotated_minimized_2020-06-29_18-13-03.sky.zip2020-06-29 15:20:4490961922,76490
BSA-QC_68fmol_Jun2020_Impact-II_minPanorama_2020-06-28_22-05-23.sky.zip2020-06-28 22:10:23131402165
19_8_27_LARP1_Kinase_Reaction_2020-06-18_10-51-35.sky.zip2020-06-24 21:25:59264762373
Phospho_CDK2_Silencing_Experiment_Human_2020-06-23_16-21-00.sky.zip2020-06-24 21:25:5911921636
20_6_Phospho_SAMe_Combined.sky.zip2020-06-24 21:25:582,77924,31330,00390,00933
Sky0 ACE2 TMPRSS2 cell lines_2020-06-18_16-55-27.sky.zip2020-06-18 13:38:456252919732
LINCS_P100_DIA_Plate96_annotated_minimized_2020-06-11_19-02-30.sky.zip2020-06-11 16:24:3290961922,76493
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SERPINF1b_2020-06-05_16-03-56.sky.zip2020-06-11 15:34:1311262
Sky1 Synthetic SARS-CoV2 proteome library_2020-05-30_00-33-22.sky.zip2020-06-09 13:21:56121431351,2121
Sky3 52 patients nano PRM-MS2 raw_2020-05-30_00-38-21.sky.zip2020-06-09 13:20:236345734055
Sky4 5 positive patients nano PRM-MS2 refined_2020-05-30_00-52-02.sky.zip2020-06-09 13:20:22634492945
Sky6 11 positive patients micro PRM-MS2 refined_2020-05-30_00-19-04.sky.zip2020-06-09 13:11:206345527311
Sky5 91 patients micro PRM-MS2 raw_2020-05-30_00-57-41.sky.zip2020-06-09 13:11:206345527391
Sky2 Dilution series SARS Cov2 nano and microflow_2020-05-30_00-30-29.sky.zip2020-06-09 13:04:536345737118
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20191031_Cdc55AID_PRM_l2_manual3_2020-05-13_10-32-35.sky.zip2020-06-02 20:16:492627542064
20191031_Cdc55AID_PRM_l3_manual3_2020-05-13_11-25-38.sky.zip2020-06-02 20:16:492424481824
STORI_COHORT_2020-06-02_14-14-43.sky.zip2020-06-02 05:16:2773154154616102
Intra-day QC_2020-06-02_14-13-40.sky.zip2020-06-02 05:14:01731541546166
Inter-day QC_2020-06-02_14-12-41.sky.zip2020-06-02 05:13:11731541546165
Instrumental set-up QC_2020-06-02_14-10-53.sky.zip2020-06-02 05:11:30731541546165
MRM-C26-CT_2020-05-26_10-19-25.sky.zip2020-05-26 01:26:17217715461713
MRM-BaF-CT_2020-05-26_10-12-57.sky.zip2020-05-26 01:17:00217815662512
ARMeD_NEDP1_2019-11-02_13-41-15.sky.zip2020-05-25 20:17:53751481622,58918
Batch1412_2020-05-12_15-21-49.sky.zip2020-05-19 20:17:272141254
Batch1553_2020-05-12_15-28-16.sky.zip2020-05-19 20:17:272141263
Batch1508_2020-05-12_15-31-44.sky.zip2020-05-19 20:17:272151639
Batch1015_2020-05-12_15-39-40.sky.zip2020-05-19 20:17:272151631
Batch1134_2020-05-12_15-41-42.sky.zip2020-05-19 20:17:272141235
Batch1139_2020-05-12_15-45-12.sky.zip2020-05-19 20:17:272141238
Batch1039_2020-05-12_15-46-04.sky.zip2020-05-19 20:17:272141265
Batch1039b_2020-05-12_15-47-09.sky.zip2020-05-19 20:17:272141211
Batch1135_2020-05-12_15-49-04.sky.zip2020-05-19 20:17:262141264
Batch1553rep_2020-05-12_15-50-08.sky.zip2020-05-19 20:17:262141213
Batch1059_2020-05-12_15-51-24.sky.zip2020-05-19 20:17:262041284
Batch 1143_2020-05-12_15-53-25.sky.zip2020-05-19 20:17:262141265
Precision_2020-05-18_19-01-24.sky.zip2020-05-19 20:17:262141219
PRM_60min_2020-05-12_18-57-21.sky.zip2020-05-19 20:16:45417201228
PRM_9min_2020-05-18_21-07-56.sky.zip2020-05-19 20:16:44299668
PRM_9min_2020-05-19_07-53-44.sky.zip2020-05-19 20:16:10299668
LINCS_P100_DIA_Plate29_annotated_minimized_2020-05-18_18-50-11.sky.zip2020-05-18 15:59:1690961921,02896
DIAassayLibrary_Gasac-Gill_Rev2_2020-05-15_18-31-25.sky.zip2020-05-15 19:31:421,6914,7464,87926,1230
DIAassayLibrary_Gasac-Gill_Rev1_2020-05-15_17-01-05.sky.zip2020-05-15 17:38:291,5075,0745,10425,3220
Inflammatory_peptides_2020-05-13_13-35-09.sky.zip2020-05-13 10:58:1317494921860
Manually_filtered_peptides_2020-05-13_08-27-56.sky.zip2020-05-13 05:51:586253,2053,20513,12660
Prefiltering_CSF_document_2020-05-12_09-28-28.sky.zip2020-05-12 07:21:051,2225,9545,95422,91166
ResponseCurve-EGFR-LTQLGTFEDHFLSLQR_2020-01-28_09-46-19.sky.zip2020-05-11 16:35:09112434
ResponseCurve-CCND1-AYPDANLLNDR_2020-01-28_09-46-04.sky.zip2020-05-11 16:35:09112644
ResponseCurve-FOLH1-TYSVSFDSLFSAVK_2020-01-28_09-46-45.sky.zip2020-05-11 16:35:09112638
ResponseCurve-MUC1-DISEMFLQIYK_2020-01-28_09-47-16.sky.zip2020-05-11 16:35:09112428
ResponseCurve-PSA-LSEPAETDAVK_2020-01-28_09-47-42.sky.zip2020-05-11 16:35:09112644
ResponseCurve-SPINK1-QTSILIQK_2020-01-28_09-48-12.sky.zip2020-05-11 16:35:09112632
ResponseCurve-MMP9-LGLGADVAQVTGALR_2020-01-28_09-47-01.sky.zip2020-05-11 16:35:09112643
ResponseCurve-NCOA2-LLQDSSSPVDLAK_2020-01-28_09-47-28.sky.zip2020-05-11 16:35:09112442
ResponseCurve-SPRC-LEAGDHPVELLAR_2020-01-28_09-48-25.sky.zip2020-05-11 16:35:09112442
ResponseCurve-TGFB1-GGEIEGFR_2020-01-28_09-48-56.sky.zip2020-05-11 16:35:09112436
ResponseCurve-VEGFA-FMDVYQR_2020-01-28_09-49-13.sky.zip2020-05-11 16:35:09112433
ResponseCurve-CAMKK2-SLSAPGNLLTK_2020-01-28_09-45-49.sky.zip2020-05-11 16:35:09112442
ResponseCurve-ERG_pan-VIVPADPTLWSTDHVR_2020-01-28_09-46-32.sky.zip2020-05-11 16:35:09112643
ResponseCurve-SMAD4-YCQYAFDLK_2020-01-28_09-47-57.sky.zip2020-05-11 16:35:09112444
ResponseCurve-TFF3-VDCGYPHVTPK_2020-01-28_09-48-41.sky.zip2020-05-11 16:35:09112444
ResponseCurve-ANXA2-GLGTDEDSLIEIICSR_2020-01-28_09-49-31.sky.zip2020-05-11 16:35:09112441
CPDR-FFPE-ID-1193_2020-01-29_12-44-32.sky.zip2020-05-11 16:23:121616328224
CPDR-FFPE-ID-859_2020-01-29_12-41-32.sky.zip2020-05-11 16:23:121616328423
CPDR-FFPE-ID-1250_2020-01-29_12-41-18.sky.zip2020-05-11 16:23:121616328229
CPDR-FFPE-ID-1376_2020-01-29_14-56-40.sky.zip2020-05-11 16:23:121616328027
CPDR-FFPE-ID-1689_2020-01-29_12-40-48.sky.zip2020-05-11 16:23:121616328425
CPDR-FFPE-ID-636_2020-01-28_13-43-56.sky.zip2020-05-11 16:23:121616328632
CPDR-FFPE-ID-779_2020-01-28_13-41-34.sky.zip2020-05-11 16:23:121616328225
CPDR-FFPE-ID-850_2020-01-29_12-24-07.sky.zip2020-05-11 16:23:121616328223
CPDR-FFPE-ID-613_2020-01-28_13-47-05.sky.zip2020-05-11 16:23:121616328218
CPDR-FFPE-ID-623_2020-01-28_13-43-21.sky.zip2020-05-11 16:23:121616328244
CPDR-FFPE-ID-607_2020-01-28_13-42-17.sky.zip2020-05-11 16:23:121616328230
CPDR-FFPE-ID-573_2020-01-28_13-40-55.sky.zip2020-05-11 16:23:121616328415
CPDR-FFPE-ID-440_2020-01-28_13-27-00.sky.zip2020-05-11 16:23:121616328021
CPDR-FFPE-ID-497_2020-01-28_11-16-43.sky.zip2020-05-11 16:23:121616328430
CPDR-FFPE-ID-428_2020-01-28_11-16-26.sky.zip2020-05-11 16:23:121616328424
CPDR-FFPE-ID-1380_2020-01-29_14-57-50.sky.zip2020-05-11 16:23:121616328227
CPDR-FFPE-ID-1030_2020-01-29_14-49-38.sky.zip2020-05-11 16:23:121616328028
CPDR-FFPE-ID-726_2020-01-29_14-42-29.sky.zip2020-05-11 16:23:121616328229
CPDR-FFPE-ID-1217_2020-01-29_14-53-36.sky.zip2020-05-11 16:23:121616328224
CPDR-FFPE-ID-1059_2020-01-29_14-51-26.sky.zip2020-05-11 16:23:121616328425
CPDR-FFPE-ID-837_2020-01-29_14-45-59.sky.zip2020-05-11 16:23:121616328422
CPDR-FFPE-ID-835_2020-01-29_14-45-16.sky.zip2020-05-11 16:23:111616328428
CPDR-FFPE-ID-675_2020-01-29_14-30-10.sky.zip2020-05-11 16:23:111616328428
CPDR-FFPE-ID-659_2020-01-29_13-41-39.sky.zip2020-05-11 16:23:111616328224
CPDR-FFPE-ID-808_2020-01-29_12-41-02.sky.zip2020-05-11 16:23:111616328235
CPDR-FFPE-ID-1442_2020-01-29_12-40-36.sky.zip2020-05-11 16:23:111616328422
PknG physiological substrates

  • Organism: Mycobacterium bovis BCG
  • Instrument: QExactive
  • SpikeIn: No
Abstract
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 3.6.0.10162), 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 (www.uniprot.com). 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