Warscheid Lab - yeast_oxICAT

Warscheid Lab - yeast_oxICAT
Global, site-resolved, quantitative analysis of the yeast redoxome

  • Organism: Saccharomyces cerevisiae
  • Instrument: Orbitrap Elite
  • SpikeIn: No
Abstract
The generation of reactive oxygen species (ROS) is inevitably linked to life. However, the precise role of ROS in signalling and specific targets are largely unknown. We performed a global proteomic analysis to delineate the yeast redoxome to a depth of more than 4,300 unique cysteine residues in over 2,200 proteins. Mapping of redox-active thiols in proteins exposed to exogenous or endogenous mitochondria-derived oxidative stress revealed ROS-sensitive sites in several components of the translation apparatus. Mitochondria are the major source of cellular ROS. We demonstrate that increased levels of intracellular ROS caused by dysfunctional mitochondria serve as a signal to attenuate global protein synthesis. Hence, we propose a universal mechanism that controls protein synthesis by inducing reversible changes in the translation machinery upon modulating the redox status of proteins involved in translation. This crosstalk between mitochondria and protein synthesis may have an important contribution to pathologies caused by dysfunctional mitochondria.
Experiment Description
We used the quantitative thiol trapping method OxICAT (Leichert et al., 2008) to determine the oxidation status of thousands of protein thiols in wild-type yeast. For OxICAT analysis upon peroxide stress strain YPH499 was grown in three biological replicates at 28°C on minimal synthetic medium containing 2% galactose to OD600 about 0.6 and treated or not with 1 mM H2O2 for 30 min. For OxICAT analysis of the temperature-sensitive Mia mutant mia40-4int and corresponding wild type (YPH499) strains were grown in four biological replicates at 19°C on minimal synthetic medium containing 3% [v/v] glycerol supplemented with 0.05-0.2% [v/v] glucose. Mutant and control cultures were shifted for 3 h to restrictive temperature (37°C). OD600 prior harvesting reached 0.8-1. For mitochondria isolation, the yeast strains YPH499 or BY4741 were grown on YPG medium (1% [w/v] yeast extract, 2% [w/v] peptone, 3% [v/v] glycerol) or YPGal medium (1% [w/v] yeast extract, 2% [w/v] peptone, 2% [v/v] galactose). Yeast cells or isolated mitochondria were pelleted, suspended in 10% TCA and immediately frozen in liquid N2. Protein extracts were prepared by bead beating in 10% TCA. Proteins were denatured in the presence of 13C-ICAT to label reduced cysteine residues. Reversibly oxidized cysteine residues were reduced by TCEP and labelled by 12C-ICAT. Proteins were digested by trypsin and ICAT labelled peptides were enriched by streptavidin affinity chromatography. Nano-HPLC-ESI-MS/MS analyses were performed on an Orbitrap Elite mass spectrometer (Thermo Scientific. OxICAT labeled samples from whole protein extracts were run twice as a technical replicate. Mass spectrometric data of OxICAT samples were processed by MaxQuant (v.1.4.1.2)(Cox and Mann, 2008) for peptide identification. MS1 ion chromatograms were extracted using Skyline (v.2.5.0)(MacLean et al., 2010) for selected peptides. Graphical displays of all chromatographic traces were visually inspected for correct peak picking. Integrated peak areas of the heavy and light version of the peptide ions were exported and the proportion of reversibly oxidized cysteine residues (%oxidation) was calculated.
Sample Description
Please note: Our data sets are from discovery-type proteomics experiments: The mass spectrometer was operated in data dependent acquisition (DDA) mode. Data is only stored on PanoramaWeb as a "Targeted MS Experiment" because it is the only option available. The whole protein extract datasets contain two Skyline documents for each experiment ("6_20" contains peptides with a length from 6 to 21 amino acids and "21_44" contains peptides between 21 and 44 aa. For OxICAT samples of isolated mitochondria there is an additional Skyline document. In the datasets of whole protein extracts the three biological replicates of the untreated (control) samples are named U1, U2, U3, the three biological replicates of the corresponding H2O2 treated samples are named H2O2_1, H2O2_2, H2O2_3, the four biologcal replicates of the mutant mia40-4int are named Mia1, Mia2, Mia3, Mia4 and the four biological replicates of wildtype control samples are named WT1, WT2, WT3, WT4 respectively. The endings "_TR1" or "_TR2" stand for technical replicate 1 or 2 respectively. The MitoDataset contains the data of (untreated) isolated mitochondria. "BY" stands for the strain BY4741 and "YPH" for the strain YPH499.
Created on 1/23/18, 5:10 PM


Site-resolved, large-scale analysis of the yeast redoxome. Yeast cells grown in 2% galactose medium were immediately frozen in 10% TCA (1). Extracted proteins were denatured in 6 M urea in the presence of heavy ICAT (13C-ICAT) to label free thiol groups (2). Reversibly oxidized cysteine residues were reduced by TCEP and labeled by light ICAT (12C-ICAT) (3). Proteins were digested with trypsin and ICAT-labeled peptides were enriched by streptavidin affinity chromatography (4). Following cleavage of the biotin tag, peptides were analyzed in duplicate by LC-MS/MS. (b) For the accurate determination of the redox state of cysteine residues, peptides were identified based on fragment ions observed in MS2 spectra using MaxQuant (v.1.4.1.2) and then quantified by extracting MS1 ion chromatograms using Skyline (v.2.5.0). Following integration of peak areas of heavy and light peptide variants, the proportion of reversibly oxidized cysteine residues (% oxidation) was calculated.

Please note: When the retention time profiles of “light” and “heavy” peaks were inconsistent, the respective XICs were excluded from quantification. The information which XICs were not quantified can be found in the tables below (denoted by “nq”).

Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
LINCS_P100_DIA_Plate63a_annotated_minimized_2019-11-14_15-28-26.sky.zip2020-02-0790961922,76494
LINCS_P100_DIA_Plate34_annotated_minimized_2019-08-30_17-25-21.sky.zip2020-02-07909619275296
LINCS_P100_PRM_Plate28_annotated_minimized_2018-05-02_18-51-02.sky.zip2020-02-0790961921,15896
LINCS_P100_PRM_Plate25_annotated_minimized_2018-05-02_18-24-10.sky.zip2020-02-0790961921,24296
LINCS_P100_DIA_Plate61_annotated_minimized_2018-02-06_17-09-06.sky.zip2020-02-07909619299095
LINCS_P100_DIA_Plate59a_annotated_minimized_2018-11-30_14-13-58.sky.zip2020-02-0790961922,76494
LINCS_P100_DIA_Plate33_annotated_minimized_2018-05-02_17-47-07.sky.zip2020-02-07909619279696
LINCS_P100_DIA_Plate23_annotated_minimized_2018-05-02_18-13-55.sky.zip2020-02-07909619296096
LINCS_P100_DIA_Plate20_annotated_minimized_2018-05-02_21-04-33.sky.zip2020-02-0790961921,07496
LINCS_P100_DIA_Plate65_annotated_minimized_2019-10-21_16-54-32.sky.zip2020-02-0790961921,58694
LINCS_P100_DIA_Plate22_annotated_minimized_2018-05-02_22-33-32.sky.zip2020-02-0790961921,09695
LINCS_P100_DIA_Plate19_annotated_minimized_2018-05-02_17-18-22.sky.zip2020-02-0790961921,16096
LINCS_P100_DIA_Plate30_annotated_minimized_2018-05-02_18-33-32.sky.zip2020-02-07909619278596
LINCS_P100_PRM_Plate32_annotated_minimized_2018-05-02_20-45-26.sky.zip2020-02-0790961921,14696
LINCS_P100_DIA_Plate67_annotated_minimized_2018-08-02_13-56-24.sky.zip2020-02-0790961922,75496
LINCS_P100_DIA_Plate66_annotated_minimized_2018-08-31_10-36-13.sky.zip2020-02-0790961922,76496
LINCS_P100_PRM_Plate31_annotated_minimized_2018-05-02_18-00-17.sky.zip2020-02-07909619299684
LINCS_P100_PRM_Plate29_03H_annotated_minimized_2018-05-02_21-16-28.sky.zip2020-02-0790961921,53696
LINCS_P100_DIA_Plate60_annotated_minimized_2017-11-29_16-22-26.sky.zip2020-02-07909619297895
LINCS_P100_DIA_Plate58_annotated_minimized_2017-11-28_11-50-08.sky.zip2020-02-07909619283495
LINCS_P100_DIA_Plate24_annotated_minimized_2018-05-02_19-07-44.sky.zip2020-02-07909619273296
LINCS_P100_PRM_Plate18_annotated_minimized_2020-01-08_17-11-19.sky.zip2020-02-0790961921,53896
LINCS_P100_DIA_Plate62_annotated_minimized_2018-04-20_15-55-05.sky.zip2020-02-07909619298696
LINCS_P100_DIA_Plate64_annotated_minimized_2018-05-02_20-34-19.sky.zip2020-02-07909619298295
LINCS_P100_DIA_Plate27_annotated_minimized_2018-05-02_19-16-47.sky.zip2020-02-0790961921,06496
LINCS_P100_DIA_Plate16_annotated_minimized_2018-05-02_19-43-10.sky.zip2020-02-0790961921,12295
LINCS_P100_DIA_Plate17_annotated_minimized_2018-05-02_17-35-29.sky.zip2020-02-0790961921,05495
LINCS_P100_DIA_Plate15_annotated_minimized_2018-05-02_19-32-22.sky.zip2020-02-0790961921,20296
LINCS_GCP_Plate59_annotated_minimized_2019-09-26_16-33-28.sky.zip2020-02-07818116279095
LINCS_GCP_Plate34_annotated_minimized_2019-10-17_13-06-51.sky.zip2020-02-07606012051896
LINCS_GCP_Plate19_annotated_minimized_2018-05-07_14-42-13.sky.zip2020-02-07606012052296
LINCS_GCP_Plate64_annotated_minimized_2018-04-20_14-41-08.sky.zip2020-02-07818116283289
LINCS_GCP_Plate61_annotated_minimized_2018-01-10_11-34-56.sky.zip2020-02-07818116276996
LINCS_GCP_Plate60_annotated_minimized_2018-04-20_14-36-43.sky.zip2020-02-07818116287696
LINCS_GCP_Plate32_annotated_minimized_2018-05-07_15-51-20.sky.zip2020-02-07606012051696
LINCS_GCP_Plate27_annotated_minimized_2018-05-07_14-51-21.sky.zip2020-02-07606012049296
LINCS_GCP_Plate23_annotated_minimized_2018-05-07_14-31-53.sky.zip2020-02-07595911850296
LINCS_GCP_Plate20_annotated_minimized_2018-05-07_15-41-44.sky.zip2020-02-07606012051496
LINCS_GCP_Plate18_annotated_minimized_2018-05-07_14-22-16.sky.zip2020-02-07606012050696
LINCS_GCP_Plate16_annotated_minimized_2018-05-09_11-16-44.sky.zip2020-02-07595911851696
LINCS_GCP_Plate22_annotated_minimized_2018-05-15_14-16-43.sky.zip2020-02-07606012050693
LINCS_GCP_Plate66_annotated_minimized_2018-09-05_11-44-04.sky.zip2020-02-07818116284896
LINCS_GCP_Plate62_annotated_minimized_2018-04-20_14-38-35.sky.zip2020-02-07818116286696
LINCS_GCP_Plate58_annotated_minimized_2018-01-02_14-26-56.sky.zip2020-02-07818116286896
LINCS_GCP_Plate31_annotated_minimized_2018-05-07_16-53-24.sky.zip2020-02-07606012051896
LINCS_GCP_Plate67_annotated_minimized_2018-07-18_16-39-05.sky.zip2020-02-07818116291496
LINCS_GCP_Plate33_annotated_minimized_2018-05-07_15-20-54.sky.zip2020-02-07606012050496
LINCS_GCP_Plate15_annotated_minimized_2018-05-02_22-46-29.sky.zip2020-02-07606012053296
LINCS_GCP_Plate65_annotated_minimized_2019-10-17_15-57-42.sky.zip2020-02-07818116282496
LINCS_GCP_Plate63_annotated_minimized_2019-09-12_11-57-23.sky.zip2020-02-07818116288296
LINCS_GCP_Plate17_annotated_minimized_2018-05-07_14-10-50.sky.zip2020-02-07606012051696
LINCS_GCP_Plate30_annotated_minimized_2018-05-07_16-32-39.sky.zip2020-02-07606012050696
LINCS_GCP_Plate29_annotated_minimized_2018-05-15_13-51-04.sky.zip2020-02-07606012051695
LINCS_GCP_Plate28_annotated_minimized_2018-05-07_15-02-14.sky.zip2020-02-07606012050696
LINCS_GCP_Plate25_annotated_minimized_2018-05-07_16-09-52.sky.zip2020-02-07616112252896
LINCS_GCP_Plate24_annotated_minimized_2018-05-07_16-43-23.sky.zip2020-02-07595911848493
matchedmatrixcalcurve_yeastyeast_2020-01-23.sky.zip2020-01-293,38527,46127,461126,57842
HuPl_5donors_Mediators_SN_2019-07-24_15-27-24.sky.zip2020-01-29150244225
HuPl_5donors_Mediators_pellet_2019-07-24_15-25-22.sky.zip2020-01-29150244225
PRM_scheduled_HumanPlatelet_5donors_5conditions_2019-07-24_15-23-54.sky.zip2020-01-291105334025
HuPl_DIA_Validation_2020-01-29_08-50-26.sky.zip2020-01-29701410414
LINCS_P100_PRM_Plate18_annotated_minimized_2020-01-08_17-11-19.sky.zip2020-01-2990961921,53896
Stabilitytest_2020-01-24_15-11-30.sky.zip2020-01-24257215848030
PRM_MS_vs_OND_2020-01-24_14-50-15.sky.zip2020-01-2424448827912
Digestion_test_Set1_2019-07-04_14-14-53.sky.zip2020-01-24246412837415
Digestion_test_Set3_2019-07-04_14-26-58.sky.zip2020-01-24246412837415
Digestion_test_Set2_2019-07-04_14-20-27.sky.zip2020-01-24246412837415
Responsecurves_rep3_2019-07-05_11-25-08.sky.zip2020-01-2415173410213
Responsecurves_rep1_2019-07-05_11-21-39.sky.zip2020-01-2415173410213
Responsecurves_rep2_2019-07-05_11-22-28.sky.zip2020-01-2415173410212
QEPlus_WideWindowDIA_2020-01-22_14-59-08.sky.zip2020-01-231052932932,0681
QEPlus_NarrowWindowDIA_2020-01-22_15-07-40.sky.zip2020-01-231544844846,1311
Ludwig_Ecoli_PQP_Absolute_AQUA_Ribo_Seq_2018-05-03_14-04-22.sky.zip2020-01-223039723667
20150514_Plasma_remove_points_LKP_curated.sky.zip2020-01-1518848455912
LINCS_P100_DIA_Plate91_Broad.sky.zip2020-01-0990961922,76454
LINCS_P100_DIA_Plate91_Pweb.sky.zip2020-01-0990961922,76454
LINCS_P100_PRM_Plate18_annotated_minimized_2020-01-08_17-11-19.sky.zip2020-01-0890961921,53896
LINCS_GCP_Plate83_annotated_minimized_2020-01-06_14-56-50.sky.zip2020-01-06818116291458
LINCS_P100_PRM_PlateAD1_annotated_minimized_2019-12-20_18-30-35.sky.zip2019-12-2090961921,33296
PRM_7x5mix_A40010_QEHF_examples_v3.sky.zip2019-12-2017352506
Arbacia session 2 2019-09-17_10-05-34.sky.zip2019-12-1919501002006
Arbacia session 1 2019-09-18_07-49-30.sky.zip2019-12-1919501002009
LINCS_GCP_Plate70_annotated_minimized_metadataupdate_2019-12-16_17-22-40.sky.zip2019-12-16818116280285
LINCS_GCP_Plate72_annotated_minimized_2019-12-16_16-53-04.sky.zip2019-12-16818116280291
20191014_LRH_GW_lumos_wide_2019-12-12_13-31-06.sky.zip2019-12-121731,6261,6268,2534
20191014_LRH_GW_lumos_newiRT_2019-12-12_11-19-52.sky.zip2019-12-121691,4071,4077,0461
190208_MW_SaKi_phospho_trypsin_new_2019-04-05_14-00-47.sky.zip2019-12-11413141108
190830_MW_MiWr_p_LysC_pub.sky.zip2019-12-11111818
190830_MW_MiWr_p_trypsin_pub.sky.zip2019-12-111332418
190208_MW_SaKi_phospho_LysC_new_2019-04-05_11-39-07.sky.zip2019-12-11124328
LINCS_P100_DIA_Plate69_annotated_minimized_2019-12-11_10-54-09.sky.zip2019-12-1190961922,76296
LINCS_P100_DIA_Plate71c_annotated_minimized_2019-12-09_11-18-17.sky.zip2019-12-0990961922,75811
LINCS_GCP_Plate65_annotated_minimized_2019-10-17_15-57-42.sky.zip2019-11-22818116282496
LINCS_GCP_Plate63_annotated_minimized_2019-09-12_11-57-23.sky.zip2019-11-22818116288296
LINCS_GCP_Plate59_annotated_minimized_2019-09-26_16-33-28.sky.zip2019-11-22818116279095
LINCS_GCP_Plate34_annotated_minimized_2019-10-17_13-06-51.sky.zip2019-11-22606012051896
LINCS_P100_DIA_Plate65_annotated_minimized_2019-10-21_16-54-32.sky.zip2019-11-2290961921,58694
LINCS_P100_DIA_Plate63a_annotated_minimized_2019-11-14_15-28-26.sky.zip2019-11-2290961922,76494
LINCS_P100_DIA_Plate34_annotated_minimized_2019-08-30_17-25-21.sky.zip2019-11-22909619275296
20190712_Bx7_fuerPanorama_2019-09-05_13-44-07.sky.zip2019-11-192222613

Table S2. Oxidation status of cysteine containing peptides of non-stressed yeast cells.  MS1 ion chromatograms were extracted using Skyline (v.2.5.0). Integrated peak areas of the “heavy” (13C-ICAT labelled) and “light” (12C-ICAT labelled) version of the peptide ions were exported and the proportion of reversibly oxidized (12C-ICAT labelled) cysteine residues (%oxidation) was calculated.  The table contains all cysteine containing peptide sequences quantified in Skyline in at least two of three biological replicates.

  Attached Files  
   
 S2_PeptideQuantification_BasalRedoxome_v4.xlsx

Table S3. Peptides identified and quantified in oxICAT labeled isolated mitochondria. The table contains all cys containing peptide sequences identified with posterior error probability (PEP) < 0.01 and intensity >0 by MaxQuant search. Reverse entries were removed.

  Attached Files  
   
 S3_MitoIso_v5.xlsx

Table S5. Oxidation status of cysteine containing peptides of yeast cells upon H2O2 stress. This table contains all cysteine containing peptide sequences quantified in Skyline in ≥  2 biological replicates of H2O2 treated samples and ≥  2 biological replicates of control samples.

  Attached Files  
   
 S5_PeptideQuantification_H2O2Treatment_v5.xlsx

Peptides identified in OxICAT experiments of mia40-4int and wildtype control samples. This table contains all cysteine-containing peptide sequences identified in mia40-4int samples wildtype control samples with posterior error probability (PEP) < 0.01 and intensity > 0 and all non-cysteine containing peptide sequences identified with PEP<0.01. It includes quantification data for all peptide sequences quantified in Skyline in ≥  3 biological replicates of mia40-4int samples and in ≥  3 biological replicates of wildtype control.

  Attached Files  
   
 SupplementaryTable7_v01.xlsx