Metadata |
datasetIdentifier | PASS00489 |
datasetType | MSMS |
submitter | Ulrike Kusebauch <ukusebauch@systemsbiology.org> |
submitter_organization | Institute for Systems Biology |
lab_head_full_name | Robert L. Moritz |
lab_head_email | Robert.Moritz@systemsbiology.org |
lab_head_organization | Institute for Systems Biology |
lab_head_country | United States |
datasetTag | Mtb_pTyr |
datasetTitle | Mycobacterium tuberculosis supports protein tyrosine phosphorylation |
publicReleaseDate | 2014-06-12 00:00:00 |
finalizedDate | 2014-06-12 20:29:59 |
summary | Reversible 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. |
contributors | Ulrike Kusebauch, Corrie Ortega, Anja Ollodart, Richard S. Rogers, David R. Sherman, Robert L. Moritz, and Christoph Grundner |
publication | Kusebauch, 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 |
growth | Mtb 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 | |
digestion | Tryptic 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. |
acquisition | LC-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. |
informatics | Data 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. |
instruments | Thermo Scientific LTQ-Velos Orbitrap, Thermo Scientific LTQ-Velos Pro Orbitrap Elite,Thermo Scientific Q Exactive |
species | Mycobacterium tuberculosis |
massModifications | static: C+57.021464; variable: M+15.9949; S+79.966331; T+79.966331; Y+79.966331 |