Schilling - MRM_HR_TripleTOF_MS2_FullScanFiltering

Schilling - MRM_HR_TripleTOF_MS2_FullScanFiltering

Multiplexed, scheduled high resolution sMRM-HR acquisition on full scan MS/MS instruments integrating data dependent and targeted, quantitative mass spectrometric workflows.

All Acquisitions were performed on a TripleTOF 5600 mass spectrometer:

 

Data sets uploaded to Panorama:

Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
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
Multiplexed, scheduled high resolution (sMRM-HR) acquisition on a full scan QqTOF instrument with integrated data-dependent and targeted mass spectrometric workflows.

  • Organism: B. taurus, S. cerevisiae, E. coli
  • Instrument: TripleTOF 5600
  • SpikeIn: No
Abstract
Faster scanning capabilities of high resolution mass spectrometers have expanded their functionality beyond data-dependent acquisition (DDA) to targeted proteomics with higher precision. Transitioning from discovery workflows to targeted peptide quantitation assays on a single high resolution LC-MS system provides an opportunity to rapidly developing targeted assays with high multiplexing by taking advantage of retention time scheduling. We therefore investigated the feasibility of implementing highly multiplexed peptide quantitation assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR) derived from discovery data sets on a single orthogonal quadrupole time-of flight (QqTOF) TripleTOF 5600 LC-MS system. We assessed the selectivity and reproducibility of MRM-HR, also referred to as parallel reaction monitoring (PRM), by measuring standard peptide concentration curves and system suitability assays. Evaluating up to 500 peptides per LC-MS run, the robustness and accuracy of MRM-HR assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and mass accuracy of full scan MS/MS spectra resulted in sufficient selectivity to monitor 6-10 MS/MS fragment ions per precursor ion and provided flexibility for post-acquisition assay refinement and optimization. We demonstrate the applicability of this workflow to complex biological samples in a yeast lysate repeatability study monitoring 532 precursor ions, and by quantitatively profiling 466 peptide precursor ions in whole cell lysates from wild-type and mutant E. coli strains with sMRM-HR to validate a previously generated candidate list of differentially expressed proteins. These results establish a robust sMRM-HR workflow to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation.
Experiment Description
MRM-HR and scheduled sMRM-HR experiments acquired on a TripleTOF 5600 (SCIEX). i) MRM-HR Response curve of 6 Protein Mix in complex matrix (C. elegans lysate), acquisition of 3 reproducibility test injections, and 2 response curve replicates; ii) MRM-HR System Suitability Study using 6 Protein Mix (10 replicate acquisitions, 3x blanks); iii) scheduled sMRM-HR Reproducibility study of proteins from yeast whole cell lysate (3 replicates); iv) scheduled sMRM-HR - Differential expression of proteins comparing E. coli wild type and ackA mutant strains (3 replicates each for WT and ackA mutant).
Sample Description
i) Response curve for spiked digested six protein mix in complex matrix (C. elegans whole cell lysate). A mixture of six pre-digested proteins (‘six protein mix’) was spiked into digested C. elegans whole cell lysate (1 ug on column) at 8 concentrations points spanning from 15 attomoles to 62.5 femtomoles (blank, 0.015, 0.061, 0.244, 0.975, 3.9, 15.6, and 62.5 fmol). Two replicate concentration curves, each with injections from lowest to highest spike concentration were acquired on the TripleTOF 5600 (MRM-HR mode). ii) Predigested Six Protein Mix was purchased from Michrom for the System Suitability Study following an Acquisition Protocol as described by Abbatiello et al. (Mol. Cell. Proteomics, 2014). iii) Digested whole cell lysate from yeast - reproducibility assessment and highly multiplexed scheduled sMRM-HR experiments. BY4743 yeast strain samples were grown at 30˚C in synthetic complete media, 0.67% yeast nitrogen base with ammonium sulfate (Sigma), plus required amino acids, supplemented with 2% glucose until the OD600 reached between 0.5 and 1.0. Subsequently, 1x1e08 cells were harvested, washed twice with water, pelleted, and frozen. Cell pellets were defrosted and re-suspended in 100 µl yeast lysis buffer (25 mM HEPES pH 7.5, 5 mM MgCl2, 50 mM KCl, 10% glycerol, Complete Mini Protease Inhibitors (Roche) and 1 volume acid-washed beads. Cells were lysed with three 1 min cycles of beating and icing using a Biospec Mini Beadbeater-8. Cell lysate were separated from beads, transferred to a new tube, and centrifuged at 15,800 g for 5 min at 4 ˚C. Finally, the supernatant was drawn off and used for downstream proteomic analysis. Samples were suspended and denatured in a final solution of 6 M urea, 100 mM Tris and tryptic digestion was performed. iv) Whole cell lysates from E. coli mutant (ackA) and WT strains – scheduled MRM-HR, differential protein expression. Briefly, E. coli WT and isogenic mutant strains cells were grown at 37˚C in TB7 [1% (w/v) tryptone buffered at pH 7.0 with potassium phosphate (100 mM)] supplemented with 0.4% glucose. Cell pellets were suspended in 6 mL of PBS and centrifuged at 4 °C, 15,000 g for 20 min. The cell pellet was collected, re-suspended, and denatured in a final solution of 6 M urea, 100 mM Tris, 75 mM NaCl. Samples were sonicated on ice (5x each), cellular debris removed, and the supernatant of each sample further processed for tryptic digestion.
Created on 7/17/15, 4:00 PM