Decker Lab - STAT1, STAT2, IRF9 BioIDs

Decker Lab - STAT1, STAT2, IRF9 BioIDs
Clustergrammer Heatmap
Download 20:20:303,67930,17230,172149,42516 20:20:303,02413,84713,84768,58624 20:20:303,02413,84713,84768,58616 20:20:303,02413,84713,84768,58620 14:33:331,2156,5256,52547,07469 21:49:252202016942 17:22:221261625 12:31:12211116014 12:31:12211116022 16:41:512,43912,24812,24873,4884 13:03:152,63513,53513,53581,2104 12:56:452,58014,16014,16084,9604 12:48:102,72514,88814,888105,89512
Smoke_Inhalation_BALF_(+) 17:36:551101881,18523
Smoke_Inhalation_BALF_(-) 17:36:471302722,22025
Smoke_Inhalation_Plasma_(+) 17:36:331102271,38922
Smoke_Inhalation_Plasma_(-) 17:36:241403352,81822
Plasma_Lipid_Library_(-) 17:35:571503713,1323
Plasma_Lipid_Library_(+) 17:35:461304462,7983 08:37:351281912
HIV-1 infant cohort for p24pep assay 21:03:012231526 21:03:012231810
HIV-1 adult cohort for p24pep assay 21:03:012231556
CFP-10pep result for six HIV-1 and TB co-infected 21:03:011121014 21:03:012231810
p24pep standard curve using recombinant 21:03:011121021
p24pep assay using HIV-1 seroconversion panel of 21:03:011121025
p24pep result for six HIV-1 and TB co-infected 21:03:012231513
VP40 p12 21:03:013674227
Other species PBS with 21:03:01366369
p24pep assay result for assay sensitivity and 21:03:012231517
MRM analysis of two VP40 peptides for standard curve with 21:03:012453061
WUSTL IDG 47 Peptide Set 18:46:1933479428245
WUSTL IDG 2 Peptide Set 18:46:082241245
WUSTL IDG 17 Peptide Set 18:46:0511173410245
WUSTL IDG 47 Peptide Set 18:30:583347942821
WUSTL IDG 2 Peptide Set 18:30:50224121
WUSTL IDG 17 Peptide Set 18:30:461117341021 20:44:231321863761,57215 20:44:2375911827086 20:43:251602274581,90639 20:42:101562274641,88216 20:42:10729018270616 20:40:01688316664830 20:40:011492184481,80830 20:38:57688717467610 20:38:578111223088210 16:33:19131402166 17:34:301488092 17:34:3060636693492 18:47:003110310347155
Calibration peptide 20:18:281121033
Calibration, peptide 20:18:281121254
Calibration peptide 20:18:28112630
Calibration peptide 20:18:28112824
Calibration peptide 20:18:28112830
Calibration, peptide 20:18:281121026
Calibration peptide 20:18:281121030
Calibration peptide 20:18:281121027
Calibration peptide 20:18:281121430
Calibration peptide 20:18:281121024
Proteomic analysis of 134 newborn 20:18:289102093134
Trypsin digestion time 20:18:28910209321
Matrix effect on protein 20:18:28910209821
Initial screening_SRMatlas 12:40:4011313651
Initial screening_Skyline 12:40:4012424751
Quantitative performance of SIL-TCT and SIL-ExC5 dissolved in water and SDC 12:40:40110209612
SiL-Ex peptides solubility 12:40:40110209636
Incubation time optimization-additional measurement for SIL-ExC3N3 peptide 12:40:401121018
Optimization of incubation time for trypsin 12:40:39110209672
Comparison of all types of internal 12:40:39110209615 12:40:39110209636
SPE 12:40:3911020966 13:59:2490565620 13:59:2490888850 08:07:143030606018 06:10:36328371724 08:33:26325911884730 08:33:26325911884125 08:33:26377014054226 08:33:26325911884715
20180731_UPR_ARoos_H& 07:57:138214214228
Nrxn3 HA IP splice segment analysis2021-07-13 08:24:0947147011
Nrxn3 AS5 knock-out analysis2021-07-13 08:17:391153216631
Nrxn3 Exon 25 Titration Curve2021-07-13 08:15:0518169214
Figure 09:08:069203410230
Figure 09:08:069203410222
Figure 09:08:059203410227
Figure 09:08:059203410227
Figure 09:08:059203410222
Figure 09:08:059203410222 07:13:4711245
PRM Splice Assays2021-06-24 08:36:0739841659900
DDR2_response 08:35:5527469264434
210429to210501_ApoE_5x5_Precision.sky2021-06-22 12:52:48151024139 08:22:184510801 13:23:364510801 14:04:322,75014,30914,30985,8544 13:37:032,78715,39815,39892,3884
DDR2_response 11:52:4827469264434
Homeostatic and Interferon-induced gene expression represent different states of promoter-associated transcription factor ISGF3
ProteomeXchange: PXD013251
  • Organism: Mus musculus
  • Instrument: Q Exactive HF-X
  • SpikeIn: No
  • Keywords: Targeted MS, PRM, quantification, BioID
  • Submitter: Thomas Gossenreiter
Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity. Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Mechanisms to meet this demand on the relevant molecular machinery include remodeling of chromatin but also changes in transcription factor interaction prior and during the IFN response. Our results show how distinct transcription factor complexes, determine the responsiveness of Interferon stimulated genes to different IRF9-containing complexes. Raw 264.7 macrophages expressing a doxycycline-inducible, myc-tagged versions of each IRF9-BirA*, STAT2-BirA* and STAT1-BirA* fusion genes were used to study complex formation in vivo. Furthermore, we extended the BioID proximity labeling by coupling it to parallel reaction monitoring to determine the degree and quantity of association between IRF9 and STATs in resting and interferon-treated macrophages.
Experiment Description
PRM assays were generated based on shotgun MS measurements acquired with the same MS instrument, selecting up to 10 high intensity proteotypic peptides for STAT1, STAT2, STAT3 and IRF9, with no missed cleavages, no methionine, and an even distribution over the chromatographic gradient. PRM assay generation was performed using Skyline. After a test run with a pooled sample showing high target protein expression, we reduced the targets to at least 5 peptides with a single charge state per protein by further optimising for best signal-to-noise and an even distribution over the gradient, resulting in a scheduled PRM assay with 4 min windows. Samples were spiked with 100 fmol Pierce Peptide Retention Time Calibration Mixture (PRTC, Thermo-Fisher) to monitor the chromatographic and nano-spray stability across the PRM measurements of all samples. Data analysis, manual validation of all peptides and their transitions (based on retention time, relative ion intensities, and mass accuracy), and relative quantification was performed in Skyline. The most intense non-interfering transition(s) of the top 5 peptides per protein were selected and their peak areas were summed up for peptide quantification (total peak area). Missing peptide intensities were imputed by random values derived from a normal distribution (down shift: median -2.15* standard dev., width: standard dev. *0.05). To correct for minor varying sample injection amounts and instrument stability over the measurements, MS1 signals of 6 stable background proteins were extracted selected based on an ANOVA analysis (q-value > 0.5, log2 intensity > 25 and standard dev. < 0.2 of MaxQuant LFQ intensities of the shot-gun measurements) and used to calculate normalization factors for the PRM dataset. After normalization, peptide intensity means were calculated for protein quantification. To ascertain significant interactions, mean log2 protein intensity ratios, standard deviation and t-test statistics were calculated for each of the target proteins.
Sample Description
BioID was performed according to a published protocol in three independent biological replicates per construct and condition. mycBioID was a gift from Kyle Roux (Addgene plasmid 35700). 5 x 106 stable Raw 264.7 cells were seeded on 15 cm dishes and treated with 0.2 µg/ml doxycycline for 24h. 50 µM biotin was added for 18 additional hours. Cells were either left untreated or stimulated for 2 h with IFN-beta or IFN-gamma or for 18 h with IFN-beta. Untreated controls were harvested at corresponding time points. Cells were washed and lysed at room temperature (lysis Buffer: 50 mMTris pH7.4; NaCL 500 mM; 0.2% SDS; EDTA 5mM + 1x protease inhibitors). Triton X-100 and 50 mMTris pH7.4 were added and the protein lysates were sonicated 2x for 30 seconds. Lysates were centrifuged for 5 minutes at full speed and supernatant were transferred to a new tube. Magnetic Pierce Streptavidin beads #88817 were washed 3x with lysis buffer. 105 µl beads were incubated with 1.3 mg of protein lysate over night at 4°C. 21 µl beads were kept for western blot analysis, the rest of the beads was used for the analysis with liquid chromatography mass spectrometry. Beads were washed at room temperature with wash buffer 1(2% SDS in H2O), wash buffer 2 (0.1% deoxycholic acid; 1% TritonX-100, 1mM EDTA, 500 mM NaCl, 50 mM HEPES; H2O) and wash buffer 3 (0.5% deoxycholic acid; 0,5% NP-40; 1 mM EDTA; 250 mM LiCl, 10 mM Tris pH7.4;H2O). Beads were washed 5 times with 50 mM Tris pH 7.4 and another two times with 50 mM ammonium bicarbonate (ABC) and then resuspended in 24 µL of 1 M urea in 50 mM ABC. 10 mM dithiothreitol (DTT) was added and the samples were incubated for 30 min at room temperature before adding 20 mM iodoacetamide and incubating for another 30 min at room temperature in the dark. Remaining iodoacetamide was quenched by adding 5 mM DTT and the proteins were digested with 300 ng (Trypsin Gold, Promega) at 37°C overnight. After stopping the digest by addition of 0.5% trifluoroacetic acid (TFA), and washing the beads with 30 µL 0.1% TFA, the supernatants were were loaded onto C18 stagetips to desalt the peptides prior to LC-MS. Peptides were separated on an Ultimate 3000 RSLC nano-flow chromatography system (Thermo-Fisher), using a pre-column for sample loading (Acclaim PepMap C18, 2 cm × 0.1 mm, 5 μm, Thermo-Fisher), and a C18 analytical column (Acclaim PepMap C18, 50 cm × 0.75 mm, 2 μm, Thermo-Fisher), applying a segmented linear gradient from 2% to 35% solvent B (80% acetonitrile, 0.1% formic acid; solvent A 0.1% formic acid) at a flow rate of 230 nL/min over 60 minutes. Eluting peptides were analyzed on a Q Exactive HF-X Orbitrap mass spectrometer (Thermo-Fisher), which was coupled to the column with a customized nano-spray EASY-Spray ion-source (Thermo-Fisher) using coated emitter tips (New Objective). For PRM data acquisition we used following MS parameters: survey scan with 60k resolution, AGC 1E6, 50 ms IT, over a range of 400 to 1300 m/z, PRM scan with 30k resolution, AGC 1E5, 300 ms IT, isolation window of 0.7 m/z with 0.2 m/z offset, and NCE of 27%.
Created on 3/26/19, 8:06 AM