U. of Lleida - Yeast

U. of Lleida - Yeast
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LINCS_P100_DIA_Plate97_annotated_minimized_2020-06-29_18-13-03.sky.zip2020-06-29 15:20:4490961922,76490
BSA-QC_68fmol_Jun2020_Impact-II_minPanorama_2020-06-28_22-05-23.sky.zip2020-06-28 22:10:23131402165
19_8_27_LARP1_Kinase_Reaction_2020-06-18_10-51-35.sky.zip2020-06-24 21:25:59264762373
Phospho_CDK2_Silencing_Experiment_Human_2020-06-23_16-21-00.sky.zip2020-06-24 21:25:5911921636
20_6_Phospho_SAMe_Combined.sky.zip2020-06-24 21:25:582,77924,31330,00390,00933
Sky0 ACE2 TMPRSS2 cell lines_2020-06-18_16-55-27.sky.zip2020-06-18 13:38:456252919732
LINCS_P100_DIA_Plate96_annotated_minimized_2020-06-11_19-02-30.sky.zip2020-06-11 16:24:3290961922,76493
AMBPb_2020-06-01_14-39-27.sky.zip2020-06-11 15:34:1311261
AMBP_2020-06-01_17-29-42.sky.zip2020-06-11 15:34:13112668
AMBPc_2020-06-02_12-38-26.sky.zip2020-06-11 15:34:1311262
B2Mb_2020-06-02_15-19-17.sky.zip2020-06-11 15:34:1311268
B2M_2020-06-02_17-56-29.sky.zip2020-06-11 15:34:13112663
HBB_2020-06-03_11-25-07.sky.zip2020-06-11 15:34:13112672
IGHA1_2020-06-03_14-07-57.sky.zip2020-06-11 15:34:131121069
IGHA1b_2020-06-03_16-47-13.sky.zip2020-06-11 15:34:13112101
IGHA1c_2020-06-04_12-45-14.sky.zip2020-06-11 15:34:13112102
LYZ_2020-06-04_15-26-23.sky.zip2020-06-11 15:34:13112460
LYZb_2020-06-04_18-12-30.sky.zip2020-06-11 15:34:13112412
SERPINF1_2020-06-05_12-55-50.sky.zip2020-06-11 15:34:13112669
SERPINF1b_2020-06-05_16-03-56.sky.zip2020-06-11 15:34:1311262
Sky1 Synthetic SARS-CoV2 proteome library_2020-05-30_00-33-22.sky.zip2020-06-09 13:21:56121431351,2121
Sky3 52 patients nano PRM-MS2 raw_2020-05-30_00-38-21.sky.zip2020-06-09 13:20:236345734055
Sky4 5 positive patients nano PRM-MS2 refined_2020-05-30_00-52-02.sky.zip2020-06-09 13:20:22634492945
Sky6 11 positive patients micro PRM-MS2 refined_2020-05-30_00-19-04.sky.zip2020-06-09 13:11:206345527311
Sky5 91 patients micro PRM-MS2 raw_2020-05-30_00-57-41.sky.zip2020-06-09 13:11:206345527391
Sky2 Dilution series SARS Cov2 nano and microflow_2020-05-30_00-30-29.sky.zip2020-06-09 13:04:536345737118
20191031_Cdc55AID_PRM_l1_manual3_2020-05-02_10-59-22.sky.zip2020-06-02 20:16:492828562164
20191031_Cdc55AID_PRM_l2_manual3_2020-05-13_10-32-35.sky.zip2020-06-02 20:16:492627542064
20191031_Cdc55AID_PRM_l3_manual3_2020-05-13_11-25-38.sky.zip2020-06-02 20:16:492424481824
STORI_COHORT_2020-06-02_14-14-43.sky.zip2020-06-02 05:16:2773154154616102
Intra-day QC_2020-06-02_14-13-40.sky.zip2020-06-02 05:14:01731541546166
Inter-day QC_2020-06-02_14-12-41.sky.zip2020-06-02 05:13:11731541546165
Instrumental set-up QC_2020-06-02_14-10-53.sky.zip2020-06-02 05:11:30731541546165
MRM-C26-CT_2020-05-26_10-19-25.sky.zip2020-05-26 01:26:17217715461713
MRM-BaF-CT_2020-05-26_10-12-57.sky.zip2020-05-26 01:17:00217815662512
ARMeD_NEDP1_2019-11-02_13-41-15.sky.zip2020-05-25 20:17:53751481622,58918
Batch1412_2020-05-12_15-21-49.sky.zip2020-05-19 20:17:272141254
Batch1553_2020-05-12_15-28-16.sky.zip2020-05-19 20:17:272141263
Batch1508_2020-05-12_15-31-44.sky.zip2020-05-19 20:17:272151639
Batch1015_2020-05-12_15-39-40.sky.zip2020-05-19 20:17:272151631
Batch1134_2020-05-12_15-41-42.sky.zip2020-05-19 20:17:272141235
Batch1139_2020-05-12_15-45-12.sky.zip2020-05-19 20:17:272141238
Batch1039_2020-05-12_15-46-04.sky.zip2020-05-19 20:17:272141265
Batch1039b_2020-05-12_15-47-09.sky.zip2020-05-19 20:17:272141211
Batch1135_2020-05-12_15-49-04.sky.zip2020-05-19 20:17:262141264
Batch1553rep_2020-05-12_15-50-08.sky.zip2020-05-19 20:17:262141213
Batch1059_2020-05-12_15-51-24.sky.zip2020-05-19 20:17:262041284
Batch 1143_2020-05-12_15-53-25.sky.zip2020-05-19 20:17:262141265
Precision_2020-05-18_19-01-24.sky.zip2020-05-19 20:17:262141219
PRM_60min_2020-05-12_18-57-21.sky.zip2020-05-19 20:16:45417201228
PRM_9min_2020-05-18_21-07-56.sky.zip2020-05-19 20:16:44299668
PRM_9min_2020-05-19_07-53-44.sky.zip2020-05-19 20:16:10299668
LINCS_P100_DIA_Plate29_annotated_minimized_2020-05-18_18-50-11.sky.zip2020-05-18 15:59:1690961921,02896
DIAassayLibrary_Gasac-Gill_Rev2_2020-05-15_18-31-25.sky.zip2020-05-15 19:31:421,6914,7464,87926,1230
DIAassayLibrary_Gasac-Gill_Rev1_2020-05-15_17-01-05.sky.zip2020-05-15 17:38:291,5075,0745,10425,3220
Inflammatory_peptides_2020-05-13_13-35-09.sky.zip2020-05-13 10:58:1317494921860
Manually_filtered_peptides_2020-05-13_08-27-56.sky.zip2020-05-13 05:51:586253,2053,20513,12660
Prefiltering_CSF_document_2020-05-12_09-28-28.sky.zip2020-05-12 07:21:051,2225,9545,95422,91166
ResponseCurve-EGFR-LTQLGTFEDHFLSLQR_2020-01-28_09-46-19.sky.zip2020-05-11 16:35:09112434
ResponseCurve-CCND1-AYPDANLLNDR_2020-01-28_09-46-04.sky.zip2020-05-11 16:35:09112644
ResponseCurve-FOLH1-TYSVSFDSLFSAVK_2020-01-28_09-46-45.sky.zip2020-05-11 16:35:09112638
ResponseCurve-MUC1-DISEMFLQIYK_2020-01-28_09-47-16.sky.zip2020-05-11 16:35:09112428
ResponseCurve-PSA-LSEPAETDAVK_2020-01-28_09-47-42.sky.zip2020-05-11 16:35:09112644
ResponseCurve-SPINK1-QTSILIQK_2020-01-28_09-48-12.sky.zip2020-05-11 16:35:09112632
ResponseCurve-MMP9-LGLGADVAQVTGALR_2020-01-28_09-47-01.sky.zip2020-05-11 16:35:09112643
ResponseCurve-NCOA2-LLQDSSSPVDLAK_2020-01-28_09-47-28.sky.zip2020-05-11 16:35:09112442
ResponseCurve-SPRC-LEAGDHPVELLAR_2020-01-28_09-48-25.sky.zip2020-05-11 16:35:09112442
ResponseCurve-TGFB1-GGEIEGFR_2020-01-28_09-48-56.sky.zip2020-05-11 16:35:09112436
ResponseCurve-VEGFA-FMDVYQR_2020-01-28_09-49-13.sky.zip2020-05-11 16:35:09112433
ResponseCurve-CAMKK2-SLSAPGNLLTK_2020-01-28_09-45-49.sky.zip2020-05-11 16:35:09112442
ResponseCurve-ERG_pan-VIVPADPTLWSTDHVR_2020-01-28_09-46-32.sky.zip2020-05-11 16:35:09112643
ResponseCurve-SMAD4-YCQYAFDLK_2020-01-28_09-47-57.sky.zip2020-05-11 16:35:09112444
ResponseCurve-TFF3-VDCGYPHVTPK_2020-01-28_09-48-41.sky.zip2020-05-11 16:35:09112444
ResponseCurve-ANXA2-GLGTDEDSLIEIICSR_2020-01-28_09-49-31.sky.zip2020-05-11 16:35:09112441
CPDR-FFPE-ID-1193_2020-01-29_12-44-32.sky.zip2020-05-11 16:23:121616328224
CPDR-FFPE-ID-859_2020-01-29_12-41-32.sky.zip2020-05-11 16:23:121616328423
CPDR-FFPE-ID-1250_2020-01-29_12-41-18.sky.zip2020-05-11 16:23:121616328229
CPDR-FFPE-ID-1376_2020-01-29_14-56-40.sky.zip2020-05-11 16:23:121616328027
CPDR-FFPE-ID-1689_2020-01-29_12-40-48.sky.zip2020-05-11 16:23:121616328425
CPDR-FFPE-ID-636_2020-01-28_13-43-56.sky.zip2020-05-11 16:23:121616328632
CPDR-FFPE-ID-779_2020-01-28_13-41-34.sky.zip2020-05-11 16:23:121616328225
CPDR-FFPE-ID-850_2020-01-29_12-24-07.sky.zip2020-05-11 16:23:121616328223
CPDR-FFPE-ID-613_2020-01-28_13-47-05.sky.zip2020-05-11 16:23:121616328218
CPDR-FFPE-ID-623_2020-01-28_13-43-21.sky.zip2020-05-11 16:23:121616328244
CPDR-FFPE-ID-607_2020-01-28_13-42-17.sky.zip2020-05-11 16:23:121616328230
CPDR-FFPE-ID-573_2020-01-28_13-40-55.sky.zip2020-05-11 16:23:121616328415
CPDR-FFPE-ID-440_2020-01-28_13-27-00.sky.zip2020-05-11 16:23:121616328021
CPDR-FFPE-ID-497_2020-01-28_11-16-43.sky.zip2020-05-11 16:23:121616328430
CPDR-FFPE-ID-428_2020-01-28_11-16-26.sky.zip2020-05-11 16:23:121616328424
CPDR-FFPE-ID-1380_2020-01-29_14-57-50.sky.zip2020-05-11 16:23:121616328227
CPDR-FFPE-ID-1030_2020-01-29_14-49-38.sky.zip2020-05-11 16:23:121616328028
CPDR-FFPE-ID-726_2020-01-29_14-42-29.sky.zip2020-05-11 16:23:121616328229
CPDR-FFPE-ID-1217_2020-01-29_14-53-36.sky.zip2020-05-11 16:23:121616328224
CPDR-FFPE-ID-1059_2020-01-29_14-51-26.sky.zip2020-05-11 16:23:121616328425
CPDR-FFPE-ID-837_2020-01-29_14-45-59.sky.zip2020-05-11 16:23:121616328422
CPDR-FFPE-ID-835_2020-01-29_14-45-16.sky.zip2020-05-11 16:23:111616328428
CPDR-FFPE-ID-675_2020-01-29_14-30-10.sky.zip2020-05-11 16:23:111616328428
CPDR-FFPE-ID-659_2020-01-29_13-41-39.sky.zip2020-05-11 16:23:111616328224
CPDR-FFPE-ID-808_2020-01-29_12-41-02.sky.zip2020-05-11 16:23:111616328235
CPDR-FFPE-ID-1442_2020-01-29_12-40-36.sky.zip2020-05-11 16:23:111616328422
Nitric oxide prevents Aft1 activation and metabolic remodeling in Frataxin-deficient yeast

  • Organism: Saccharomyces cerevisiae
  • Instrument: Agilent 6420 Triple Quadrupole Spectrometer
  • SpikeIn: Yes
Abstract
Yeast frataxin homolog (Yfh1) is the orthologue of human frataxin, a mitochondrial protein whose deficiency causes Friedreich Ataxia. Yfh1 deficiency activates Aft1, a transcription factor governing iron homeostasis in yeast cells. Although the mechanisms causing this activation are not completely understood, it is assumed that it may be caused by iron-sulfur deficiency. However, several evidences indicate that activation of Aft1 occurs in the absence of iron-sulfur deficiency. Besides, Yfh1 deficiency also leads to metabolic remodeling (mainly consisting in a shift from respiratory to fermentative metabolism) and to induction of Yhb1, a nitric oxide (NO) detoxifying enzyme. In this work, we have used conditional Yfh1 mutant yeast strains to investigate the relationship between NO, Aft1 activation and metabolic remodeling. We have observed that NO prevents Aft1 activation caused by Yfh1 deficiency. This phenomenon is not observed when Aft1 is activated by iron scarcity or impaired iron-sulfur biogenesis. In addition, analyzing key metabolic proteins by a targeted proteomics approach, we have observed that NO prevents the metabolic remodeling caused by Yfh1 deficiency. We conclude that Aft1 activation in Yfh1-deficient yeasts is not caused by iron-sulfur deficiency or iron scarcity. Our hypothesis is that Yfh1 deficiency leads to the presence of anomalous iron species that can compromise iron bioavailability and activate a signaling cascade that results in Aft1 activation and metabolic remodeling.
Experiment Description
Yeast cells were resuspended in 50 mM Tris-HCl buffer pH 7,5 plus 1 mM EDTA and a mixture of protease inhibitors and were disrupted using glass beads. After, SDS was added to a final concentration of 2% and protein extracts were vortexed, boiled and centrifuged (12000 rpm 10 min). Proteins were quantified using the Lowry assay and 30 ug were precipitated with cold acetone (9 volumes) and resuspended in 8M urea, 0.1M ammonium bicarbonate. Then, proteins were subjected to reduction by 12 mM DTT and alquilation by 40mM iodoacetamide. Samples were diluted with 0.1 M ammonium bicarbonate to a final concentration of 1.5 M urea and sequencing grade porcine trypsin (Promega) was added to a final enzyme:substrate ratio of 1:100. After digestion, 0,8 ul from a heavy peptide standards mixture was added to the sample. The approximate concentration of each heavy peptide in this mixture was 3,5 uM. Heavy peptides were obtained from JPT (SpikeTidesTM_L). The resulting peptide mix was purified and enriched using C18 columns (Pierce C-18 Spin Columns, Thermo Scientific). Eluted fractions from the C18 column were evaporated using a Concentrator Plus (Eppendorf) and peptides were resuspended in 5% acetonitrile plus 0,1% formic acid. All peptide samples were analyzed on a triple quadrupole spectrometer (Agilent 6420) equipped with an electrospray ion source. Chromatographic separations of peptides were performed on an Agilent 1200 LC system using a Supelco Bioshell A160 Peptide C18 column (1 mm x 15 cm). Peptides (up to 15 micrograms of protein digest) were separated with a linear gradient of acetonitrile/water, containing 0.1% formic acid, at a flow rate of 75 ul/min. A gradient from 5 to 60% acetonitrile in 60 minutes was used. The mass spectrometer was operated in multiple reaction monitoring mode. Transitions were obtained from SRM atlas and imported into Skyline software (MacLean et al, 2010). Each MRM acquisition was performed with Q1 and Q3 operated at unit resolution (0.7 m/z half maximum peak width). Once validated and optimized, the SRM assays were used to quantify all the analyzed peptides using scheduled SRM mode in a single run (retention time window, 120 s; target scan time, 2 sec).
Sample Description
Two yeast strains were used. - BQS201 (also named tetO7-YFH1), in which the YFH1 promoter is replaced by a tet- promoter. - BSS255 (also named tetO7-YFH1 deltaYHB1), which is a nul YHB1 mutant derived from BQS201. Four biological conditions were analyzed: - BQS201 (control condition) - BQS201 plus doxycycline (this drug represses YFH1 expresion). - BQS255 - BQS255 plus doxycycline Yeasts were grown in YPG and doxycycline, where indicated, added for 24 hours.
Created on 11/2/17, 1:12 PM