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A code of mono-phosphorylation modulates the function of RB

ABSTRACT: The data were acquired by performing multiplexed proteomics with TMT10-plex reagents using both and Orbitrap Fusion and an Orbitrap Lumos mass spectrometer and applying an SPS-supported LC-MS2/MS3 method. Samples were anti-FLAG immunoprecipitates of RB phospho-isoforms from human RPE1 cells. Samples were labeled with the specific TMT reagents as follows: Sample pool 1: 126, Bridge; 127n, RB wild-type (replicate 1); 127c, RB_DELTA_cdk (replicate 1); 128n, FLAG-RB wild-type (replicate 1); 128c, FLAG-RB_DELTA_cdk (replicate 1); 129n, FLAG-RB_DELTA_cdk + S230 (replicate 1); 129c, FLAG-RB_delta_cdk + S249 (replicate 1); 130n, ,FLAG-RB_delta_cdk + T252 (replicate 1); 131, Bridge. Sample pool 2: 126, Bridge; 127n, RB wild-type (replicate 2); 127c, RB_delta_cdk (replicate 2); 128n, FLAG-RB wild-type (replicate 2); 128c, FLAG-RB_delta_cdk (replicate 2); 129n, FLAG-RB_delta_cdk + S230 (replicate 2); 129c, FLAG-RB_delta_cdk + S249 (replicate 2), 130n, ,FLAG-RB_delta_cdk + T252 (replicate 2), 131, Bridge. Sample pool 3: 126, Bridge; 127n, RB wild-type (replicate 3); 127c, RB_delta_cdk (replicate 3); 128n, FLAG-RB wild-type (replicate 3); 128c, FLAG-RB_delta_cdk (replicate 3); 129n, FLAG-RB_delta_cdk + S230 (replicate 3); 129c, FLAG-RB_delta_cdk + S249 (replicate 3); 130n, FLAG-RB_delta_cdk + T252 (replicate 3); 131, Bridge. Sample pool 4: 126, Bridge; 127n, FLAG-RB_delta_cdk + T356 (replicate 1); 127c, FLAG-RB_delta_cdk + T373 (replicate 1); 128n, FLAG-RB_delta_cdk + S608 (replicate 1); 128c, FLAG-RB_delta_cdk + S612 (replicate 1); 129n, FLAG-RB_delta_cdk + S780 (replicate 1); 129c, FLAG-RB_delta_cdk + S788 (replicate 1); 130n, FLAG-RB_delta_cdk + S795 (replicate 1); 131, Bridge. Sample pool 5: 126, Bridge; 127n, FLAG-RB_delta_cdk + T356 (replicate 2); 127c, FLAG-RB_delta_cdk + T373 (replicate 2); 128n, FLAG-RB_delta_cdk + S608 (replicate 2); 128c, FLAG-RB_delta_cdk + S612 (replicate 2); 129n, FLAG-RB_delta_cdk + S780 (replicate 2); 129c, FLAG-RB_delta_cdk + S788 (replicate 2); 130n, FLAG-RB_delta_cdk + S795 (replicate 2); 131, Bridge. Sample pool 6: 126, Bridge; 127n, FLAG-RB_delta_cdk + T356 (replicate 3); 127c, FLAG-RB_delta_cdk + T373 (replicate 3); 128n, FLAG-RB_delta_cdk + S608 (replicate 3); 128c, FLAG-RB_delta_cdk + S612 (replicate 3); 129n, FLAG-RB_delta_cdk + S780 (replicate 3); 129c, FLAG-RB_delta_cdk + S788 (replicate 3); 130n, FLAG-RB_delta_cdk + S795 (replicate 3); 131, Bridge. Sample pool 7: 126, Bridge; 127n, FLAG-RB_delta_cdk + S807 (replicate 1); 127c, FLAG-RB_delta_cdk + S811 (replicate 1); 128n, FLAG-RB_delta_cdk + T821 (replicate 1); 128c, FLAG-RB_delta_cdk + T826 (replicate 1); 131, Bridge. Sample pool 8: 126, Bridge; 127n, FLAG-RB_delta_cdk + S807 (replicate 2); 127c, FLAG-RB_delta_cdk + S811 (replicate 2); 128n, FLAG-RB_delta_cdk + T821 (replicate 2); 128c, FLAG-RB_delta_cdk + T826 (replicate 2); 131, Bridge. Sample pool 9: 126, Bridge; 127n, FLAG-RB_delta_cdk + S807 (replicate 3); 127c, FLAG-RB_delta_cdk + S811 (replicate 3); 128n, FLAG-RB_delta_cdk + T821 (replicate 3); 128c, FLAG-RB_delta_cdk + T826 (replicate 3); 131, Bridge.

INSTRUMENT(S): Orbitrap Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Nicholas Dyson

PROVIDER: MSV000082562 | MassIVE | Sun Jul 01 10:19:00 BST 2018

REPOSITORIES: MassIVE

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Project description:We used mass spectrometry-based proteomics to perform a phosphoproteomics analysis of selinexor-treated ex vivo AML patient samples (n=20) and 4 AML cell lines (PL-21, NOMO-1, GDM-1 and MV4-11). For quantitative phosphoproteomics, we used a tandem mass tag (TMT)-based approach. Conditions for the TMT 11-plex (Ex vivo samples) and TMT 8-plex (AML cell lines) setups are specified below. Cells were treated with 1 M selinexor (also known as KPT-330) or 0.1% DMSO for 6 hours. The experimental treatment conditions and TMT11-plex (ex vivo samples) and TMT8-plex (cell lines) labeling schemes are specified below: TMT-11-plex TMT channel Patient no Treatment Set 1 126 Ex_24 DMSO Set 1 127N Ex_24 Selinexor Set 1 127C Ex_25 DMSO Set 1 128N Ex_25 Selinexor Set 1 128C Ex_34 DMSO Set 1 129N Ex_34 Selinexor Set 1 129C Ex_41 DMSO Set 1 130N Ex_41 Selinexor Set 1 130C Ex_44 DMSO Set 1 131N Ex_44 Selinexor Set 1 131C Mix pool Mix pool Set 2 126 Ex_9 DMSO Set 2 127N Ex_9 Selinexor Set 2 127C Ex_18 DMSO Set 2 128N Ex_18 Selinexor Set 2 128C Ex_27 DMSO Set 2 129N Ex_27 Selinexor Set 2 129C Ex_29 DMSO Set 2 130N Ex_29 Selinexor Set 2 130C Ex_36 DMSO Set 2 131N Ex_36 Selinexor Set 2 131C Mix pool Mix pool Set 3 126 Ex_8 DMSO Set 3 127N Ex_8 Selinexor Set 3 127C Ex_13 DMSO Set 3 128N Ex_13 Selinexor Set 3 128C Ex_14 DMSO Set 3 129N Ex_14 Selinexor Set 3 129C Ex_19 DMSO Set 3 130N Ex_19 Selinexor Set 3 130C Ex_40 DMSO Set 3 131N Ex_40 Selinexor Set 3 131C Mix pool Mix pool Set 4 126 Ex_6 DMSO Set 4 127N Ex_6 Selinexor Set 4 127C Ex_15 DMSO Set 4 128N Ex_15 Selinexor Set 4 128C Ex_28 DMSO Set 4 129N Ex_28 Selinexor Set 4 129C Ex_39 DMSO Set 4 130N Ex_39 Selinexor Set 4 130C Ex_42 DMSO Set 4 131N Ex_42 Selinexor Set 4 131C Mix pool Mix pool AML cell lines: TMT-8-plex TMT channel Treatment Replicate GDM-1 126 DMSO 1 GDM-1 127N DMSO 2 GDM-1 127C DMSO 3 GDM-1 128N DMSO 4 GDM-1 128C Selinexor 1 GDM-1 129N Selinexor 2 GDM-1 129C Selinexor 3 GDM-1 130N Selinexor 4 MV4-11 126 DMSO 1 MV4-11 127N DMSO 2 MV4-11 127C DMSO 3 MV4-11 128N DMSO 4 MV4-11 128C Selinexor 1 MV4-11 129N Selinexor 2 MV4-11 129C Selinexor 3 MV4-11 130N Selinexor 4 NOMO-1 126 DMSO 1 NOMO-1 127N DMSO 2 NOMO-1 127C DMSO 3 NOMO-1 128N DMSO 4 NOMO-1 128C Selinexor 1 NOMO-1 129N Selinexor 2 NOMO-1 129C Selinexor 3 NOMO-1 130N Selinexor 4 PL-21 126 DMSO 1 PL-21 127N DMSO 2 PL-21 127C DMSO 3 PL-21 128N DMSO 4 PL-21 128C Selinexor 1 PL-21 129N Selinexor 2 PL-21 129C Selinexor 3 PL-21 130N Selinexor 4

Project description:Proteome maps of biochemically enriched synaptic tissue from human Alzheimers Disease (AD) and control angular gyrus samples to highlight the synapse as a site of integration for pathophysiological processes active in AD. The samples were combined into eleven TMTpools (01 to 11). Samples pools were fractionated using basic pH reversed-phase chromatography (bRPLC) and at least 12 fractions were analyzed by LC-MS2/MS3 per pool. The RAW files are: Carlyle_TMTpool_01_fraction_01 to Carlyle_TMTpool_01_fraction_12 Carlyle_TMTpool_02_fraction_01 to Carlyle_TMTpool_02_fraction_12 Carlyle_TMTpool_03_fraction_01 to Carlyle_TMTpool_03_fraction_12 Carlyle_TMTpool_04_fraction_01 to Carlyle_TMTpool_04_fraction_12 Carlyle_TMTpool_05_fraction_01 to Carlyle_TMTpool_05_fraction_12 Carlyle_TMTpool_06_fraction_01 to Carlyle_TMTpool_06_fraction_12 Carlyle_TMTpool_07_fraction_01 to Carlyle_TMTpool_07_fraction_12 Carlyle_TMTpool_08_fraction_01 to Carlyle_TMTpool_08_fraction_08, Carlyle_TMTpool_08_fraction_09A, Carlyle_TMTpool_08_fraction_09B, Carlyle_TMTpool_08_fraction_10 to Carlyle_TMTpool_08_fraction_12 Carlyle_TMTpool_09_fraction_01 to Carlyle_TMTpool_09_fraction_12 Carlyle_TMTpool_10_fraction_01 to Carlyle_TMTpool_10_fraction_12 Carlyle_TMTpool_11_fraction_01 to Carlyle_TMTpool_11_fraction_12 The labeling scheme is: TMTpool_01: RUSH_0592 (126), RUSH_0034 (127n), RUSH_0755 (127c), RUSH_0143 (128n), RUSH_1442 (128c), RUSH_0570 (129n), Pooled_sample (129c), Pooled_sample (130n), RUSH_0992 (130c), RUSH_0857 (131n), bridge (131c). TMTpool_02: RUSH_0974 (126), RUSH_1440 (127n), RUSH_0240 (127c), RUSH_0032 (128n), RUSH_0406 (128c), RUSH_0065 (129n), RUSH_1376 (129c), RUSH_0684 (130n), RUSH_0269 (130c), RUSH_1159 (131n), bridge (131c). TMTpool_03: RUSH_0136 (126), Pooled_sample (127n), RUSH_0537 (127c), RUSH_1250 (128n), RUSH_0795 (128c), RUSH_0001 (129n), RUSH_0404 (129c), RUSH_0606 (130n), RUSH_0229 (130c), RUSH_0465 (131n), bridge (131c). TMTpool_04: RUSH_0734 (126), RUSH_0873 (127n), RUSH_0743 (127c), Pooled_sample (128n), RUSH_0712 (128c), RUSH_0751 (129n), RUSH_0302 (129c), RUSH_0499 (130n), RUSH_1218 (130c), RUSH_1421 (131n), bridge (131c). TMTpool_05: RUSH_1392 (126), RUSH_0890 (127n), RUSH_1006 (127c), RUSH_1063 (128n), RUSH_0128 (128c), RUSH_0590 (129n), RUSH_0674 (129c), RUSH_0572 (130n), RUSH_0413 (130c), RUSH_1288 (131n), bridge (131c). TMTpool_06: RUSH_1487 (126), RUSH_1348 (127n), RUSH_0007 (127c), RUSH_1323 (128n), RUSH_1498 (128c), RUSH_0976 (129n), RUSH_0859 (129c), RUSH_0544 (130n), RUSH_0067 (130c), RUSH_1384 (131n), bridge (131c). TMTpool_07: RUSH_0212 (126), RUSH_1474 (127n), Pooled_sample (127c), RUSH_0010 (128n), RUSH_0931 (128c), RUSH_1205 (129n), RUSH_1200 (129c), RUSH_0206 (130n), RUSH_0376 (130c), RUSH_0460 (131n), bridge (131c). TMTpool_08: RUSH_0927 (126), RUSH_0963 (127n), RUSH_0055 (127c), RUSH_0085 (128n), RUSH_0780 (128c), RUSH_0327 (129n), RUSH_0354 (129c), RUSH_0123 (130n), RUSH_1358 (130c), RUSH_1424 (131n), bridge (131c). TMTpool_09: RUSH_1334 (126), RUSH_0921 (127n), Pooled_sample (127c), RUSH_1093 (128n), RUSH_1499 (128c), RUSH_1419 (129n), Pooled_sample (129c), RUSH_0555 (130n), RUSH_0618 (130c), RUSH_0732 (131n), bridge (131c). TMTpool_10: RUSH_1176 (126), Pooled_sample (127n), RUSH_0958 (127c), RUSH_0447 (128n), RUSH_1485 (128c), RUSH_0158 (129n), RUSH_0252 (129c), RUSH_0078 (130n), RUSH_0415 (130c), Pooled_sample (131n), bridge (131c). TMTpool_11: RUSH_1313 (126), RUSH_0989 (127n), RUSH_0295 (127c), RUSH_0177 (128n), RUSH_0064 (128c), RUSH_0919 (129n), RUSH_0847 (129c), RUSH_0266 (130n), RUSH_0933 (130c), Pooled_sample (131n), bridge (131c).

Project description:Mass spectrometry-based proteomics was applied to unravel Erk signaling in mouse embryonic stem cells (ESCs). A previously described engineered ESCs system for Erk induction via a drug-inducible cRAF-ErT2 fusion (Hamilton, W. B. & Brickman, J. M., Cell Rep 2014) was used in two different setups measuring the phosphoproteome in an 11-plex tandem mass tag (TMT)-based approach as described below. Fgf4 stimulation and MEK inhibition: ESCs were stimulated with Fgf4 (40 nM, recombinant, mouse, 5 minutes) following pretreatment (30 minutes) with either DMSO (1:10000) or MEK inhibition (MEKi: 1 microM PD0325901): The experimental treatment conditions and TMT11-plex labeling are specified below: 126: DMSO, unstimulated control replicate 1 127N: DMSO, unstimulated control replicate 2 127C: DMSO, unstimulated control replicate 3 128N: DMSO + Fgf4 replicate 1 128C: DMSO + Fgf4 replicate 2 129N: DMSO + Fgf4 replicate 3 129C: MEKi + Fgf4 replicate 1 130N: MEKi + Fgf4 replicate 2 130C: MEKi + Fgf4 replicate 3 131N: MEKi replicate 1 131C: MEKi replicate 2 Analog sensitive Erk2 setup: In the ESC system the ERK2-/- ESCs (Hamilton et al., PloS ONE, 2013) were engineered to stably express an inducible cRAF-ErT2 fusion protein expressed from the same transgene as a FLAG-tagged analogue sensitive Erk2Q103A. Erk1 was subsequently removed by CRISPR-Cas9 mutagenesis using sgRNAs flanking exon 2. Cells were pre-treated for 30 minutes with either the ATP-mimetic compound 1-NM-PP1 (2 microM) (Endo et al., Biochem Biophys Res Commun 2006), the Mek1 inhibitor PD0325901 (1 microM), or DMSO (0.01%), followed by induction or not of the cRAF-ErT2 fusion by (Z)-4-Hydroxytamoxifen (4OHT) (250 nM) for 2 hours as indicated. The experimental treatment conditions and TMT11-plex labeling are specified below: 126: DMSO pretreatment, non-induced control, replicate 1 127N: DMSO pretreatment, non-induced control, replicate 2 127C: DMSO pretreatment, non-induced control, replicate 3 128N: DMSO pretreatment, 4OHT-induction, replicate 1 128C: DMSO pretreatment, 4OHT-induction, replicate 2 129N: DMSO pretreatment, 4OHT-induction, replicate 3 129C: PP1 pretreatment, 4OHT-induction, replicate 1 130N: PP1 pretreatment, 4OHT-induction, replicate 2 130C: PP1 pretreatment, 4OHT-induction, replicate 3 131N: MEKi pretreatment, 4OHT-induction, replicate 1 131C: MEKi pretreatment, 4OHT-induction, replicate 2

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A code of mono-phosphorylation modulates the function of RB

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