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Dataset Identifier

Metadata
datasetIdentifierPASS00489
datasetTypeMSMS
submitterUlrike Kusebauch <ukusebauch@systemsbiology.org>
submitter_organizationInstitute for Systems Biology
lab_head_full_nameRobert L. Moritz
lab_head_emailRobert.Moritz@systemsbiology.org
lab_head_organizationInstitute for Systems Biology
lab_head_countryUnited States
datasetTagMtb_pTyr
datasetTitleMycobacterium tuberculosis supports protein tyrosine phosphorylation
publicReleaseDate2014-06-12 00:00:00
finalizedDate2014-06-12 20:29:59
summaryReversible protein phosphorylation determines growth and adaptive
decisions in Mycobacterium tuberculosis (Mtb). At least 11
two-component systems and 11 Ser/Thr protein kinases (STPKs)
mediate phosphorylation on Asp, His, Ser, and Thr. In contrast,
protein phosphorylation on Tyr has not been described previously
in Mtb. Here, using a combination of phospho-enrichment and
highly sensitive mass spectrometry, we show extensive protein
Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several
STPKs function as dual-specificity kinases that phosphorylate
Tyr in cis and in trans, suggesting that dual-specificity kinases have
a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine
site of the essential STPK PknB reduces its activity in
vitro and in live Mtb, indicating that Tyr phosphorylation has
a functional role in bacterial growth. These data identify a previously
unrecognized phosphorylation system in a human pathogen
that claims ∼1.4 million lives every year.
contributorsUlrike Kusebauch, Corrie Ortega, Anja Ollodart, Richard S. Rogers, David R. Sherman, Robert L. Moritz, and Christoph Grundner
publicationKusebauch, U, Ortega, C, Ollodart, A, Rogers, RS, Sherman,DR, Moritz, RL,Grundner, C,Mycobacterium tuberculosis supports protein tyrosine phosphorylation, PNAS 2014 ; published ahead of print June 9, 2014, doi:10.1073/pnas.1323894111
growthMtb Strains, Culture, and Lysate Preparation. Mtb strain H37Rv (ATCC) was
grown in liquid 7H9 medium with 10% OADC in rolling cultures. The overexpressing
strains tet-pknB, tet-pknB Lsy40Ala, and tet-pknB Tyr182Phe
were generated as previously described. For cfu assays, cultures at OD600
0.05 were induced with 20ng/mL ATc and were plated daily for 7 d. For
LC-MS/MS analysis, cultures were harvested by centrifugation for 5 min at
4000 × g and were washed once in PBS. Cells were pelleted and resuspended
in 50 mM NH4HCO3/50% (vol/vol) 2,2,2-Trifluoroethanol. Cells were lysed by
bead beating and inactivated by heat killing. M. smegmatis strain MC2 155
was grown and lysate was prepared as described above for Mtb.
treatment
extraction
separation
digestionTryptic Digestion and Phosphopeptide Enrichment. Proteins were reduced with
5 mM dithiothreitol (DTT) for 30 min at 55 °C, alkylated with 14 mM
iodoacetamide (IAM) for 30 min at room temperature in darkness, followed
by quenching of unreacted IAM with 5 mM DTT. The sample was
diluted with 125 mM NH4HCO3 and digested with trypsin at an enzyme:
substrate ratio of 1:50 at 37 °C overnight. The digest was dried under centrifugal evaporation (Savant), resolubilized in 500 μL 1% trifluoroacetic acid,and peptides were desalted with tC18 SepPak cartridges (Waters). To enrich
for phosphopeptides, we performed IMAC using PHOS-Select Iron Affinity
Gel (Sigma-Aldrich) and then the Titansphere
Phos-TiO kit (GL Sciences Inc.). Before MS analysis, peptides were
desalted with a tC18 SepPak cartridge. Alternatively, Tyr-phosphorylated
proteins were purified by immunoprecipitation with a 1:1 mixture of the
anti-pTyr antibodies PY99 agarose and 4G10 Sepharose before tryptic digestion.
After three washes in lysis buffer, protein was eluted with 0.2%
trifluoroacetic acid, and samples were processed further as described above.
Recombinant proteins were reduced with 1 mM DDT, alkylated with 10 mM
IAM, and quenched with 5 mM DDT. Samples were diluted 1:1 with 125 mM
NH4HCO3 and digested with trypsin (1:50) overnight.
acquisitionLC-MS/MS Analysis. Peptides were analyzed on either an LTQ-Velos Orbitrap
or an LTQ-Velos Pro Orbitrap Elite (Thermo Fisher Scientific) mass spectrometer
equipped with a nano LC system. Peptide separation was performed
on C18 (ReproSil-Pur C18-AQ, 120 Å, 3 μm; Dr. Maisch GmbH,
Germany) capillary columns packed in house using 0.1% formic acid in
water (A) and 0.1% formic acid in acetonitrile (B) with a gradient from
3–25% (vol/vol) B in 90 min at a flow rate of 0.3 μL/min. Survey full-scan
MS spectra were acquired in the mass range m/z 400–2,000 in the Orbitrap
analyzer at a resolution of 60,000. The 10 most intense ions determined
in the survey scan were fragmented by collision-induced dissociation in
the LTQ. Dynamic exclusion was enabled. In addition, a portion of phospho-
enriched Mtb lysate was analyzed on a Q Exactive (Thermo Fisher
Scientific) by higher-energy collisional dissociation and nano LC conditions
as described above.
informaticsData Analysis. Instrument-native data files were converted to mzML or mzXML
files using the ProteoWizard msconvert program. MS/MS spectra were
associated with peptide sequences using SEQUEST (version UW2012.01.2) and
a database comprising 3,996 protein entries plus common contaminants and
a sequence-shuffled decoy counterpart. Peptides were allowed to be semitryptic
with up to two internal cleavage sites. The search parameters included
a fixed modification of +57.021464 to account for carbamidomethylated cysteines
and differential modifications of +15.9949 for oxidized methionines
and +79.966331 for phosphorylated Ser, Thr, and Tyr. The search results were
processed with the TPP (v4.6 rev1 and 4.6.2) including PeptideProphet
and iProphet. Public MS data were analyzed using the TPP with open
source SEQUEST called Comet and X!Tandem.
instrumentsThermo Scientific LTQ-Velos Orbitrap, Thermo Scientific LTQ-Velos Pro Orbitrap Elite,Thermo Scientific Q Exactive
speciesMycobacterium tuberculosis
massModificationsstatic: C+57.021464; variable: M+15.9949; S+79.966331; T+79.966331; Y+79.966331

Official URL for this dataset: http://www.peptideatlas.org/PASS/PASS00489
To access files via FTP, use credentials:
Servername: ftp.peptideatlas.org
Username: PASS00489
Password: WW5558tt

Or use your browser's FTP mode: ftp://PASS00489:WW5558tt@ftp.peptideatlas.org/


Listing of files:

 262M Jun 11  2014 EA20130223_ISBTTOMXX1707_50m_nE.raw
 552M Jun 11  2014 ELA20120524_PknH_tryptic_direct.raw
 335M Jun 11  2014 ELA20130617_PknB_digest_r01.raw
 331M Jun 11  2014 ELA20130617_PknB_digest_r02.raw
 398M Jun 11  2014 ELA20130617_PknF_digest_r01.raw
 388M Jun 11  2014 ELA20130617_PknF_digest_r02.raw
 285M Jun 11  2014 OR20110909_mut1_imac_26.RAW
 230M Jun 11  2014 OR20110909_mut1_tio2_22.RAW
 274M Jun 11  2014 OR20110909_wt1_imac_30.RAW
 259M Jun 11  2014 OR20110909_wt1_tio2_04.RAW
  469 May  2  2014 PASS00489_DESCRIPTION-2014-04-02_160031.txt
  694 May  2  2014 PASS00489_DESCRIPTION-2014-04-02_160519.txt
  822 May 22  2014 PASS00489_DESCRIPTION-2014-04-22_190612.txt
  887 Jun 12  2014 PASS00489_DESCRIPTION-2014-05-12_192836.txt
 5.7K Jun 12  2014 PASS00489_DESCRIPTION.txt
 1.4G Jun 11  2014 QE20130517_MTBpi_A1_IMAC_r01.raw
 1.3G Jun 11  2014 QE20130517_MTBpi_A1_IMAC_r02.raw
 1.3G Jun 11  2014 QE20130517_MTBpi_A1_TiO2_r01.raw
 1.3G Jun 11  2014 QE20130517_MTBpi_A1_TiO2_r01_130518203643.raw
 1.3G Jun 11  2014 QE20130517_MTBpi_A1_TiO2_r02.raw
 1.3G Jun 11  2014 QE20130517_MTBpi_A1_TiO2_r02_130518230838.raw
 1.2G Jun 11  2014 QE20130517_MTBpi_A2_IMAC_r01.raw
 1.3G Jun 11  2014 QE20130517_MTBpi_A2_IMAC_r02.raw
 467M Jun 11  2014 VE20120209_mTB_mut1_IMAC_40.raw
 458M Jun 11  2014 VE20120209_mTB_mut1_IMAC_41.raw
 444M Jun 11  2014 VE20120209_mTB_mut1_tio2_60.raw
 438M Jun 11  2014 VE20120209_mTB_mut1_tio2_61.raw
 470M Jun 11  2014 VE20120209_mTB_mut2_IMAC_43.raw
 475M Jun 11  2014 VE20120209_mTB_mut2_IMAC_44.raw
 449M Jun 11  2014 VE20120209_mTB_mut2_tio2_63.raw
 451M Jun 11  2014 VE20120209_mTB_mut2_tio2_64.raw
 470M Jun 11  2014 VE20120209_mTB_mut3_IMAC_46.raw
 463M Jun 11  2014 VE20120209_mTB_mut3_IMAC_47.raw
 449M Jun 11  2014 VE20120209_mTB_mut3_tio2_66.raw
 433M Jun 11  2014 VE20120209_mTB_mut3_tio2_67.raw
 476M Jun 11  2014 VE20120209_mTB_wt1_IMAC_30.raw
 481M Jun 11  2014 VE20120209_mTB_wt1_IMAC_31.raw
 446M Jun 11  2014 VE20120209_mTB_wt1_tio2_50.raw
 444M Jun 11  2014 VE20120209_mTB_wt1_tio2_51.raw
 477M Jun 11  2014 VE20120209_mTB_wt2_IMAC_33.raw
 489M Jun 11  2014 VE20120209_mTB_wt2_IMAC_34.raw
 457M Jun 11  2014 VE20120209_mTB_wt2_tio2_53.raw
 442M Jun 11  2014 VE20120209_mTB_wt2_tio2_54.raw
 486M Jun 11  2014 VE20120209_mTB_wt3_IMAC_36.raw
 484M Jun 11  2014 VE20120209_mTB_wt3_IMAC_37.raw
 473M Jun 11  2014 VE20120209_mTB_wt3_tio2_56.raw
 469M Jun 11  2014 VE20120209_mTB_wt3_tio2_57.raw
 1.9G Jun 11  2014 VE20120427_mTB_IMAC_04.mzML
 1.8G Jun 11  2014 VE20120427_mTB_IMAC_04B.mzML
 1.7G Jun 11  2014 VE20120427_mTB_IMAC_26.mzML
 1.7G Jun 11  2014 VE20120427_mTB_IMAC_27.mzML
 1.7G Jun 11  2014 VE20120427_mTB_IMAC_28.mzML
 1.7G Jun 11  2014 VE20120427_mTB_TiO2_05.mzML
 1.7G Jun 11  2014 VE20120427_mTB_TiO2_05B.mzML
 1.5G Jun 11  2014 VE20120427_mTB_TiO2_42.mzML
 1.6G Jun 11  2014 VE20120427_mTB_TiO2_43.mzML
 1.6G Jun 11  2014 VE20120427_mTB_TiO2_44.mzML
 444M Jun 11  2014 VE20120501_mTB_PKnD_tryptic_direct.raw
 492M Jun 11  2014 VE20120823_PknB_digest_r01.raw
 522M Jun 11  2014 VE20120823_PknB_digest_r02.raw
 491M Jun 11  2014 VE20120823_PknE_digest_r01.raw
 500M Jun 11  2014 VE20120823_PknE_digest_r02.raw
 510M Jun 11  2014 VE20120823_PknG_digest_r01.raw
 522M Jun 11  2014 VE20120823_PknG_digest_r02.raw
 528M Jun 11  2014 VE20121119_MTB_H37Rv_S3_IMAC_01.raw
 523M Jun 11  2014 VE20121119_MTB_H37Rv_S3_IMAC_02.raw
 481M Jun 11  2014 VE20121119_MTB_H37Rv_S3_TiO2_01.raw
 467M Jun 11  2014 VE20121119_MTB_H37Rv_S3_TiO2_02.raw
 242M Jun 11  2014 VE20130215_CG_S1_r02.raw
 243M Jun 11  2014 VE20130215_CG_S1_r03.raw
  84K Jun 11  2014 overview_data_mtb.pdf

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