Muth-Pawlak - SRM_irondepr_2015

Muth-Pawlak - SRM_irondepr_2015
Clustergrammer Heatmap
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tfreedman_brian023_20190605_17363_LMWstim_2019-07-11_11-35-04.sky.zip (834 KB)2019-07-1515101006
tfreedman_brian023_20190605_17363_LMWnostim_2019-07-11_11-34-30.sky.zip (814 KB)2019-07-1515101006
tfreedman_brian023_20190605_17363_HMWstim_2019-07-11_11-34-02.sky.zip (843 KB)2019-07-1515101006
tfreedman_brian023_20190605_17363_HMWnostim_2019-07-11_11-33-22.sky.zip (834 KB)2019-07-15158806
tfreedman_brian023_20190605_17363_Calibration_2019-07-09_12-03-07.sky.zip (2 MB)2019-07-151144024
calcurve_pY32_unmodifiedY32_2019-07-09_11-44-05.sky.zip (2 MB)2019-07-151267834
Round2_S26_110515_Controls5_2019-07-08_13-48-30.sky.zip (1 MB)2019-07-0811254516814
Round2_S26_missing_Controls10_2019-07-08_13-47-57.sky.zip (431 KB)2019-07-081125451683
Round2_S26_113015_Controls9_2019-07-08_13-47-03.sky.zip (2 MB)2019-07-0811254516820
Round2_S26_111315_Controls8_2019-07-08_13-45-55.sky.zip (1 MB)2019-07-0811254516814
Round2_S26_111115_Controls7_2019-07-08_13-45-14.sky.zip (1 MB)2019-07-0811254516814
Round2_S26_110715_Controls6_2019-07-08_13-44-11.sky.zip (2 MB)2019-07-0811254516823
Round2_S26_110315_Controls4_2019-07-08_13-43-07.sky.zip (2 MB)2019-07-0811254516823
Round2_S26_110215_Controls3_2019-07-08_13-42-11.sky.zip (2 MB)2019-07-0811254516824
Round2_S26_101815_Controls2_2019-07-08_13-39-51.sky.zip (2 MB)2019-07-0811254516820
Round2_S26_033116_missing_Controls1_2019-07-08_13-38-37.sky.zip (21 MB)2019-07-081125451686
032115_S26_Results_Controls7_2019-07-08_13-30-46.sky.zip (3 MB)2019-07-0820294918224
032015_S26_Results_Controls6_2019-07-08_13-30-12.sky.zip (2 MB)2019-07-0820294918222
031715_S26_Results_Controls5_2019-07-08_13-29-40.sky.zip (2 MB)2019-07-0820294918218
031615_S26_Results_Controls4_2019-07-08_13-28-56.sky.zip (2 MB)2019-07-0820294918223
031315_S26_Results_Controls3_2019-07-08_13-28-13.sky.zip (3 MB)2019-07-0820294918230
031215_S26_Results_Controls2_2019-07-08_13-27-37.sky.zip (2 MB)2019-07-0820294918220
031115_S26_Results_Controls1_2019-07-08_13-26-51.sky.zip (2 MB)2019-07-0820294918223
CUL5_HIV_interactors_quant_2018-02-16_10-51-21.sky.zip (12 MB)2019-06-281062752751,07116
CBFB_HIV_CUL2_CUL5_interactors_quant_2018-02-16_10-59-32.sky.zip (5 MB)2019-06-2820878734118
ELOB_HIV_interactors_quant_2018-02-16_11-00-53.sky.zip (33 MB)2019-06-281906636632,63520
CBFB_HIV_interactors_quant_2018-02-16_10-55-27.sky.zip (12 MB)2019-06-28972812811,08316
Healthy_controls_round2_042617_2019-06-27_16-10-07.sky.zip (1 MB)2019-06-2710204015414
Healthy_controls_round1_042617_2019-06-27_16-09-16.sky.zip (1 MB)2019-06-2711204015620
103015_LewinsohnNegatives_round2_2019-06-27_16-02-00.sky.zip (1 MB)2019-06-2710204015415
103015_LewinsohnNegatives_round1_2019-06-27_15-31-23.sky.zip (1 MB)2019-06-2711204015615
LINCS_P100_DIA_Plate80_annotated_minimized_2019-06-13_12-13-28.sky.zip (121 MB)2019-06-1390961922,76496
LINCS_GCP_Plate71b_annotated_minimized_2019-06-10_18-12-11.sky.zip (12 MB)2019-06-10818116283018
LINCS_GCP_Plate71a_annotated_minimized_2019-06-10_18-10-31.sky.zip (5 MB)2019-06-1081811628548
LINCS_P100_DIA_Plate77_annotated_minimized_2019-06-10_16-17-36.sky.zip (81 MB)2019-06-1090961922,76266
LINCS_P100_DIA_Plate70_annotated_minimized_2019-06-03_13-47-33.sky.zip (140 MB)2019-06-0890961922,76496
LINCS_P100_DIA_Plate82_annotated_minimized_2019-06-03_16-03-24.sky.zip (110 MB)2019-06-0890961922,76491
LINCS_P100_DIA_Plate76_annotated_minimized_2019-06-07_15-29-16.sky.zip (100 MB)2019-06-0790961922,75866
LINCS_P100_QC_Plate80_annotated_minimized_2019-06-04_16-27-57.sky.zip (7 MB)2019-06-04882417496
LINCS_P100_QC_Plate82_annotated_minimized_2019-06-04_15-46-59.sky.zip (8 MB)2019-06-04882418396
LINCS_P100_QC_Plate78_annotated_minimized_2019-06-03_15-57-25.sky.zip (7 MB)2019-06-03882316196
LINCS_P100_DIA_Plate70_annotated_minimized_2019-06-03_13-47-33.sky.zip (140 MB)2019-06-0390961922,76496
LINCS_P100_QC_Plate70_annotated_minimized_2019-06-03_13-48-35.sky.zip (7 MB)2019-06-03882416596
all combined_2019-03-21_20-40-14.sky.zip (46 MB)2019-05-31133575268,5337
20180518 multiplex quantification - APOA4 only samples_2018-05-18_12-13-20.sky.zip (265 MB)2019-05-311364864
20180518 multiplex quantification only samples_2018-05-18_11-58-07.sky.zip (380 MB)2019-05-3128357050268
20180509_prest-kinetics_2018-05-09_18-52-06.sky.zip (120 MB)2019-05-311613628684820
500 µE (PG-500uE_2ul_2019-05-16_15-23-09).sky.zip (7 MB)2019-05-292710510555811
Huidan-LHCSR3-Trypsin-PRM_FINAL2_2019-05-16_17-33-35.sky.zip (3 MB)2019-05-2920611225758
Huidan-LHCSR3-LysC-selected-tSIM-PRM_FINAL_2019-05-16_17-31-57.sky.zip (13 MB)2019-05-2931681366528
200µE (R5-8_V2_2019-05-16_15-31-29).sky.zip (8 MB)2019-05-2928979751616
matchedmatrixcalcurve_yeasthela_2019-05-28_13-23-23.sky.zip (1 GB)2019-05-283,22827,28927,28985,17542
matchedmatrixcalcurve_yeastwater_2019-05-24_12-06-14.sky.zip (1 GB)2019-05-243,73136,39336,39395,63942
20150514_Plasma_remove_points_2019-05-22_18-51-25.sky.zip (162 MB)2019-05-2220989862412
matchedmatrixcalcurve_csf_2019-05-22_14-34-12.sky.zip (531 MB)2019-05-224,00716,22916,22947,85630
matchedmatrixcalcurve_yeastyeast_2019-05-22_12-49-50.sky.zip (1 GB)2019-05-223,48230,60230,60290,48842
Janschitz et al_PRM_results_Panorama upload_2019-05-06_18-03-18.sky.zip (611 MB)2019-05-094475833712
LINCS_GCP_Plate76_annotated_minimized_2019-04-29_15-46-06.sky.zip (16 MB)2019-04-29818116287833
LINCS_P100_DIA_Plate51_annotated_minimized_2017-08-02_16-21-22.sky.zip (43 MB)2019-04-26909619292496
LINCS_P100_DIA_Plate41_annotated_minimized_2017-02-16_14-31-38.sky.zip (128 MB)2019-04-2690961921,00296
LINCS_P100_DIA_PlateAD2_annotated_minimized_2019-04-18_11-17-21.sky.zip (52 MB)2019-04-18909619278892
LINCS_P100_QC_PlateAD2_annotated_minimized_2019-04-18_11-15-52.sky.zip (6 MB)2019-04-18882413292
LINCS_GCP_Plate77_annotated_minimized_2019-04-17_09-22-00.sky.zip (19 MB)2019-04-17818116291233
LINCS_GCP_Plate75_annotated_minimized_2019-04-16_13-40-09.sky.zip (26 MB)2019-04-16818116291233
LINCS_GCP_Plate74_annotated_minimized_2019-04-16_13-21-11.sky.zip (24 MB)2019-04-16808016080533
LINCS_GCP_Plate73_annotated_minimized_2019-04-16_11-45-08.sky.zip (24 MB)2019-04-16818116289433
LINCS_P100_QC_Plate75_annotated_minimized_2019-04-08_16-33-18.sky.zip (4 MB)2019-04-08882415666
LINCS_P100_DIA_Plate75_annotated_minimized_2019-04-08_16-26-05.sky.zip (72 MB)2019-04-0890961922,76466
LINCS_P100_QC_Plate76_annotated_minimized_2019-04-08_15-34-30.sky.zip (5 MB)2019-04-08882415066
Dee Dee Luu et. al_The immunogenic microbial peptide RaxX represents an unclassified group of sulfated ribosomally synthesized and post-translationally modified peptides_2019-04-02_12-44-12.sky.zip (1 MB)2019-04-062221318
LINCS_P100_DIA_Plate73_annotated_minimized_2019-04-02_12-31-00.sky.zip (75 MB)2019-04-0290961922,76466
LINCS_P100_QC_Plate74a_annotated_minimized_2019-04-01_16-25-26.sky.zip (2 MB)2019-04-01882417436
LINCS_P100_DIA_Plate74a_annotated_minimized_2019-04-01_16-19-30.sky.zip (39 MB)2019-04-0190961922,76236
20181129_HFF1mut_IP_PRM_2019-03-15_14-54-27.sky.zip (4 MB)2019-04-0172020935
LINCS_P100_QC_Plate73_annotated_minimized_2019-03-28_10-49-18.sky.zip (5 MB)2019-03-28882416266
LINCS_P100_QC_Plate74_annotated_minimized_2019-03-27_16-36-40.sky.zip (6 MB)2019-03-27882417182
LINCS_P100_DIA_Plate74_annotated_minimized_2019-03-27_16-18-55.sky.zip (98 MB)2019-03-2790961922,76284
PRM_final_quantification_non-normalized_2019-03-18_10-30-44.sky.zip (148 MB)2019-03-265353514748
LINCS_P100_QC_Plate70_annotated_minimized_2019-03-13_14-12-46.sky.zip (7 MB)2019-03-13882416596
HLF with ladder_GUnames_2018-08-15_19-59-17.sky.zip (176 KB)2019-03-086045451
U87MG with ladder_GUnames_2018-08-15_20-02-38.sky.zip (429 KB)2019-03-08901181181
IgA with ladder_GUnames_2018-08-15_20-00-17.sky.zip (358 KB)2019-03-08801251251
HNE with ladder_GUnames_2018-08-15_19-59-46.sky.zip (174 KB)2019-03-086038381
PGM OG with ladder_GUnames_2018-08-15_20-01-34.sky.zip (370 KB)2019-03-08201061061
IgG with ladder_GUnames_2018-08-15_20-01-05.sky.zip (228 KB)2019-03-087061611
Fet NG with ladder_GUnames_2018-08-15_19-58-42.sky.zip (229 KB)2019-03-087063631
CBHI OG with ladder_GUnames_2018-08-15_19-58-20.sky.zip (93 KB)2019-03-082020201
CBHI NG with ladder_GUnames_2018-08-15_19-57-24.sky.zip (109 KB)2019-03-083028281
BLF with ladder_GUnames_2018-08-15_19-55-59.sky.zip (302 KB)2019-03-088085851
Fig3_U87MG_iRT_ladder_2018-08-15_19-50-07.sky.zip (429 KB)2019-03-08901191191
Fig5_SupFig3_Dextran_Spectral_Library_2018-08-15_19-53-02.sky.zip (40 KB)2019-03-08103181
Fig1_SupFig1_Ladder_tech_reps_2018-08-15_19-48-46.sky.zip (797 KB)2019-03-0810151510
Fig6_2019-01-26_23-48-44.sky.zip (63 MB)2019-03-088011634212
Fig2_SupFig2_iRT_linear.sky.zip (6 MB)2019-03-0820183216
PRM-Identifying the N-terminal amino acid for a TAG start codon_2019-03-04.sky.zip (3 MB)2019-03-05266583
PRM-Identifying the N-terminal amino acid for an ATG start codon_2019-03-04.sky.zip (1 MB)2019-03-05266563
Cortex_50_targets_PRM_finaL_20181212_2019-01-15_15-55-34.sky.zip (5 MB)2019-03-01551332582,2633
Pancreas_50_targets_PRM_final_20181210_2019-01-15_15-52-49.sky.zip (5 MB)2019-03-01631342602,0613
sasp_panel_ras_2019-02-26_13-24-23.sky.zip (51 MB)2019-02-2674730,02544,411576,8660
sasp_panel_irradiation_2019-02-26_13-00-53.sky.zip (17 MB)2019-02-261729,62414,598189,6470
Development of a quantitative SRM-based proteomics method to study iron metabolism of Synechocystis sp. PCC 6803

  • Organism: Synechocystis sp. PCC 6803
  • Instrument: TSQ Vantage
  • SpikeIn: No
Abstract
The cyanobacterium Synechocystis sp. PCC 6803 is a well-established model species in oxygenic photosynthesis research, and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short and long term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short and long term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low abundant proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.
Experiment Description
The target Synechocystis sp. PCC 6803 strain was cultivated under the iron sufficient and deprived (harvested once the OD750nm reached 1.0 and after 12 days) conditions. The proteins were extracted and digested and the peptides mixtures were subjected to shotgun LC-MS/MS analysis (QExactive and Orbitrap-FT mass spectrometers). The obtained data was searched against S. 6803 proteome and the combined results led to discovery of altogether 1710 proteins, represented in total by 15031 unique peptides (46 % coverage of the S. 6803 proteome). SRM-assays were developped for 106 proteins covering three functional categories : (1) photosynthesis and carbon fixation, (2) Other anabolic reactions/ auxiliary metabolism and (3) catabolism/ amphibolic pathways. We were able to reliably detect (TSQ Vantage) and quantify 96 proteins in unfractionated cell lysates, with 87 and 92 proteins showing p-values <0.01 under short and long term iron deficiency, respectively. Moreover 64 proteins have never been quantified in similar experimental conditions.
Sample Description
Sample preparation Synechocystis sp. PCC 6803 was grown in BG11 medium, pH 8.0, at +30 °C under a light intensity of approximately 50 μmol photons m− 2 s− 1 in an AlgaeTron230 incubator under agitation of 150 rpm under 1 % (v/v) CO2. For the iron depletion the cells were grown in BG11-medium, where FeNH4-citrate was eliminated. The cell density was measured on Genesys 10S UV-VIS spectrophotometer (Thermo Scientific). The precultures were grown under normal conditions in normal BG11, pH 8.0, in an incubator filled with 1% CO2. The cell growth was monitored by measuring the optical density at 750 nm (OD750nm) over time and the cells were collected by centrifugation (4000 x g 10 minutes at room temperature) once OD750nm reached ~1.0. The cells were washed twice either with iron sufficient or depleted BG11, depending of the future treatment, and centrifuged as previously described. The washed precultures were inoculated to 50 ml batches of respective growth media and the starting OD750nm was adjusted to ~0.1. In order to remove all traces of iron from the culture flasks, the glassware were treated with 10 % HCl and 10 µM EDTA prior autoclaving. The cell growth was measured over time and the cells were harvested from both iron sufficient and deprived conditions, once the OD750nm reached 1.0 and after 12 days under iron deprivation. The growth patterns were verified by repeating the growth experiments several times. The cells were collected by centrifugationand washed twice with 50 mM TES-KOH buffer, pH 8.0. The cell pellet was suspended in protein extraction buffer (0.1 % (w/v) RapiGest SF in 8 M urea solubilized in 0.1 M NH4HCO3 supplemented with 200 µM PMSF) with equal volume of acid washed glass beads (150-212 µm, Sigma).The cells were broken by bead beater and unbroken cells and glass beads were removed by centrifugation. The proteins were reduced with dithiotreitol and alkylated with iodoacetamide. The protein extracts were precipitated with 1:5 v/v of 50% acetone/ 50% ethanol o/n at -20 °C. The pellet was solubilized by trypsin digestion (1:100 trypsin:protein ratio) in 50 mM NH4HCO3 and 5 % acetonitrile (ACN) buffer for 4-5 hours at +37 °C shaking. Digestion was continued for an additional 15-16 hours following a second addition of trypsin in the same ratio. The digestion was stopped by adding formic acid (FA) (Sigma) to a final concentration of 0.5–1 % to lower the pH below 2. The digestion mixture was incubated for 30 min at +37 °C at 130 rpm and centrifuged. To desalt the samples, solid phase extraction (SPE) of the peptide mixture was performed with 4 mm/1 ml extraction disk cartridge (Empore™ C18-SD, 3M™) according to manufactures protocol. The eluted peptide samples were vacuum-dried (Savant SPD1010, SpeedVac Concentrator, Thermo Scientific) and solubilized in 0,1 % FA and 2 % ACN and stored at -80 °C prior to MS analysis.
Created on 8/10/15, 12:58 PM