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:

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histone_garcia_v0-9-2_greer_onlyhistlibrary_2021-01-11_15-29-49.sky.zip2021-02-17 19:58:15101362386,13112
180727_H460_cisplatin_analysis_2021-02-16_12-20-31.sky.zip2021-02-16 20:32:511,48717,12017,12085,5765
OLG_PSE_PRM_2021-02-07_13-57-40.sky.zip2021-02-07 20:43:065264022029
LINCS_GCP_Plate97_annotated_minimized_2021-02-02_14-10-30.sky.zip2021-02-02 11:18:18818116286694
LINCS_GCP_Plate96_annotated_minimized_2021-02-02_13-46-59.sky.zip2021-02-02 10:51:36818116285693
BSA_QC.sky.zip2021-01-29 10:48:30166625
P155_04_Panorama_2021-01-25_16-28-55.sky.zip2021-01-27 20:07:55214147521
LINCS_GCP_Plate67_annotated_minimized_2018-07-18_16-39-05.sky.zip2021-01-23 11:44:41818116291496
BSA-QC_68fmol_Nov2020_Impact-II_2021-01-21_13-58-46.sky.zip2021-01-21 13:59:10131402166
Pilot Kinetics Experiment CVN Mice 120k_Final_2021-01-06_15-35-00.sky.zip2021-01-06 12:35:161012188
Pilot Experiment APOE3 3 Mice (21 DBS_3Plasma_2Brain)_Final_2021-01-06_15-29-12.sky.zip2021-01-06 12:29:3410162226
E4HN 69 Wk F_M_120k_Final_2021-01-06_15-27-19.sky.zip2021-01-06 12:27:5910172384
Miller-QEHF-Liver_MouseQuant-10092019_2019-10-14_12-39-29.sky.zip2020-12-24 08:41:122,78415,73615,736112,55724
Miller-QEHF-lysosomesMouseQuant-2-07152019_2019-07-15_16-47-56.sky.zip2020-12-24 08:41:113,42618,79218,792130,95824
LINCS_P100_PRM_Plate32_annotated_minimized_2018-05-02_20-45-26.sky.zip2020-12-23 22:30:1090961921,14696
LINCS_P100_PRM_Plate31_annotated_minimized_2018-05-02_18-00-17.sky.zip2020-12-23 22:00:20909619299684
LINCS_P100_DIA_Plate65_annotated_minimized_2019-10-21_16-54-32.sky.zip2020-12-23 21:58:5590961921,58694
LINCS_P100_PRM_Plate29_03H_annotated_minimized_2018-05-02_21-16-28.sky.zip2020-12-23 21:34:3590961921,53696
LINCS_P100_PRM_Plate28_annotated_minimized_2018-05-02_18-51-02.sky.zip2020-12-23 21:10:3590961921,15896
LINCS_P100_PRM_Plate18_annotated_minimized_2020-01-08_17-11-19.sky.zip2020-12-23 20:45:4290961921,53896
LINCS_P100_DIA_Plate67_annotated_minimized_2018-08-02_13-56-24.sky.zip2020-12-23 20:22:5990961922,75496
LINCS_P100_PRM_Plate25_annotated_minimized_2018-05-02_18-24-10.sky.zip2020-12-23 19:57:1690961921,24296
LINCS_P100_DIA_Plate66_annotated_minimized_2018-08-31_10-36-13.sky.zip2020-12-23 19:28:3390961922,76496
LINCS_P100_DIA_Plate64_annotated_minimized_2018-05-02_20-34-19.sky.zip2020-12-23 18:45:44909619298295
LINCS_P100_DIA_Plate63a_annotated_minimized_2019-11-14_15-28-26.sky.zip2020-12-23 18:44:3190961922,76494
LINCS_P100_DIA_Plate62_annotated_minimized_2018-04-20_15-55-05.sky.zip2020-12-23 18:08:05909619298696
LINCS_P100_DIA_Plate61_annotated_minimized_2018-02-06_17-09-06.sky.zip2020-12-23 18:06:41909619299095
LINCS_P100_DIA_Plate60_annotated_minimized_2017-11-29_16-22-26.sky.zip2020-12-23 18:04:43909619297895
LINCS_P100_DIA_Plate59a_annotated_minimized_2018-11-30_14-13-58.sky.zip2020-12-23 18:02:4890961922,76494
LINCS_P100_DIA_Plate58_annotated_minimized_2017-11-28_11-50-08.sky.zip2020-12-23 17:37:47909619283495
LINCS_P100_DIA_Plate34_annotated_minimized_2019-08-30_17-25-21.sky.zip2020-12-23 17:36:23909619275296
LINCS_P100_DIA_Plate33_annotated_minimized_2018-05-02_17-47-07.sky.zip2020-12-23 17:35:27909619279696
LINCS_P100_DIA_Plate30_annotated_minimized_2018-05-02_18-33-32.sky.zip2020-12-23 17:34:11909619278596
LINCS_P100_DIA_Plate27_annotated_minimized_2018-05-02_19-16-47.sky.zip2020-12-23 17:32:2190961921,06496
LINCS_P100_DIA_Plate24_annotated_minimized_2018-05-02_19-07-44.sky.zip2020-12-23 16:42:31909619273296
LINCS_P100_DIA_Plate23_annotated_minimized_2018-05-02_18-13-55.sky.zip2020-12-23 16:41:36909619296096
LINCS_P100_DIA_Plate22_annotated_minimized_2018-05-02_22-33-32.sky.zip2020-12-23 16:40:3390961921,09695
LINCS_P100_DIA_Plate20_annotated_minimized_2018-05-02_21-04-33.sky.zip2020-12-23 16:02:0390961921,07496
LINCS_P100_DIA_Plate19_annotated_minimized_2018-05-02_17-18-22.sky.zip2020-12-23 15:42:0390961921,16096
LINCS_P100_DIA_Plate17_annotated_minimized_2018-05-02_17-35-29.sky.zip2020-12-23 15:22:0390961921,05495
LINCS_P100_DIA_Plate16_annotated_minimized_2018-05-02_19-43-10.sky.zip2020-12-23 15:01:3290961921,12295
LINCS_P100_DIA_Plate15_annotated_minimized_2018-05-02_19-32-22.sky.zip2020-12-23 13:42:2190961921,20296
LINCS_GCP_Plate66_annotated_minimized_2018-09-05_11-44-04.sky.zip2020-12-23 12:29:44818116284896
LINCS_GCP_Plate65_annotated_minimized_2019-10-17_15-57-42.sky.zip2020-12-23 12:28:15818116282496
LINCS_GCP_Plate64_annotated_minimized_2018-04-20_14-41-08.sky.zip2020-12-23 12:26:30818116283289
LINCS_GCP_Plate63_annotated_minimized_2019-09-12_11-57-23.sky.zip2020-12-23 12:24:58818116288296
LINCS_GCP_Plate62_annotated_minimized_2018-04-20_14-38-35.sky.zip2020-12-23 12:23:20818116286696
LINCS_GCP_Plate61_annotated_minimized_2018-01-10_11-34-56.sky.zip2020-12-23 12:21:57818116276996
LINCS_GCP_Plate60_annotated_minimized_2018-04-20_14-36-43.sky.zip2020-12-23 12:20:32818116287696
LINCS_GCP_Plate59_annotated_minimized_2019-09-26_16-33-28.sky.zip2020-12-23 12:19:10818116279095
LINCS_GCP_Plate58_annotated_minimized_2018-01-02_14-26-56.sky.zip2020-12-23 12:17:40818116286896
LINCS_GCP_Plate34_annotated_minimized_2019-10-17_13-06-51.sky.zip2020-12-23 12:16:10606012051896
LINCS_GCP_Plate33_annotated_minimized_2018-05-07_15-20-54.sky.zip2020-12-23 12:15:29606012050496
LINCS_GCP_Plate32_annotated_minimized_2018-05-07_15-51-20.sky.zip2020-12-23 12:14:32606012051696
LINCS_GCP_Plate31_annotated_minimized_2018-05-07_16-53-24.sky.zip2020-12-23 12:13:39606012051896
LINCS_GCP_Plate30_annotated_minimized_2018-05-07_16-32-39.sky.zip2020-12-23 12:12:51606012050696
LINCS_GCP_Plate29_annotated_minimized_2018-05-15_13-51-04.sky.zip2020-12-23 12:12:12606012051695
LINCS_GCP_Plate28_annotated_minimized_2018-05-07_15-02-14.sky.zip2020-12-23 12:11:32606012050696
LINCS_GCP_Plate27_annotated_minimized_2018-05-07_14-51-21.sky.zip2020-12-23 12:10:47606012049296
LINCS_GCP_Plate25_annotated_minimized_2018-05-07_16-09-52.sky.zip2020-12-23 12:09:55616112252896
LINCS_GCP_Plate24_annotated_minimized_2018-05-07_16-43-23.sky.zip2020-12-23 12:08:50595911848493
LINCS_GCP_Plate23_annotated_minimized_2018-05-07_14-31-53.sky.zip2020-12-23 12:07:52595911850296
LINCS_GCP_Plate22_annotated_minimized_2018-05-15_14-16-43.sky.zip2020-12-23 12:06:52606012050693
LINCS_GCP_Plate20_annotated_minimized_2018-05-07_15-41-44.sky.zip2020-12-23 12:05:51606012051496
LINCS_GCP_Plate19_annotated_minimized_2018-05-07_14-42-13.sky.zip2020-12-23 12:04:35606012052296
LINCS_GCP_Plate18_annotated_minimized_2018-05-07_14-22-16.sky.zip2020-12-23 12:03:50606012050696
LINCS_GCP_Plate17_annotated_minimized_2018-05-07_14-10-50.sky.zip2020-12-23 12:02:40606012051696
LINCS_GCP_Plate16_annotated_minimized_2018-05-09_11-16-44.sky.zip2020-12-23 12:01:46595911851696
LINCS_GCP_Plate15_annotated_minimized_2018-05-02_22-46-29.sky.zip2020-12-23 12:00:02606012053296
Proteins_PRM_Targeted_Treatment_Run_1_no_precursors_2020-09-25_09-21-58.sky.zip2020-12-18 08:02:333310310347437
Swab_host_PvsN_figure_2020-12-06_12-41-22.sky.zip2020-12-13 15:24:0010282816220
COVID_swab_viral_MRM_2020-12-11_23-25-32.sky.zip2020-12-13 15:24:005131312013
EGFR pathway peptide standards at different DDM concentrations Final_2020-11-28_19-41-16.sky.zip2020-11-30 19:49:41101010325
schilling_swath_silac-ecoli_6600_2020-10-23_12-53-04.sky.zip2020-11-22 02:16:567292,1523,03921,74141
20200828_QEHFX_lkp_pSILAC-DIA_btz_2020-10-29_10-27-57.sky.zip2020-11-22 02:16:564,08520,25840,417130,13220
20200828_QEHFX_lkp_pSILAC-DIA_curve_2020-11-04_16-10-32.sky.zip2020-11-22 02:16:566,32343,96187,681564,73484
20200828_QEHFX_lkp_pSILAC-DIA_curve_2020-10-13_09-33-04.sky.zip2020-11-22 02:16:566,32343,96187,681301,78784
20200421_ToxXevo_AZ_Delta_87.sky.zip2020-11-21 17:01:46111123489
Polyquant_Different_on_column_loading.sky.zip2020-11-21 17:01:40720376528
20200818_ToxXevo_eSwab_Comparison_Sample_Preparation.sky.zip2020-11-21 17:01:35111123327
TCA_Aceton_Digest_Time.sky.zip2020-11-21 17:01:32113143940
HCl_TCA.sky.zip2020-11-21 17:01:32113133712
MRM_XevoTQS_S63879_Patient_Samples.sky.zip2020-11-21 17:01:29110103030
MRM_XevoTQS_Dilution_series_Pure_eSwab_UTM.sky.zip2020-11-21 17:01:26110103050
Covid19_MRM_Template.sky.zip2020-11-21 17:01:23117228500
Skyline_75min_patientsamples_UZLeuven_Panorama.sky.zip2020-11-21 17:00:32112732883,37720
Leiden_eSwab_dilution.sky.zip2020-11-21 16:52:03719356210
Negative_patients.sky.zip2020-11-21 16:51:367203766105
Positive_Patients_below_Ct_30.sky.zip2020-11-21 16:51:3672037111100
200403_TTOF6600_SWATH_Dilution_20min_Panorama.sky.zip2020-11-21 16:49:38112732883,37727
200403_TTOF6600_SWATH_Dilution_75min_dilution_Panorama.sky.zip2020-11-21 16:49:38112732883,37727
Bioer_patient_dilution.sky.zip2020-11-21 16:36:23113132620
Virocult_patient_dilution.sky.zip2020-11-21 16:36:23113132621
UTM_patient_dilution.sky.zip2020-11-21 16:36:23113132621
eSwab_patient_dilution.sky.zip2020-11-21 16:36:23113132620
Lab_01.sky.zip2020-11-21 16:35:581661910
Lab_15.sky.zip2020-11-21 16:35:58616173810
Lab_14.sky.zip2020-11-21 16:35:58113142611
Lab_13.sky.zip2020-11-21 16:35:58113164911
Lab_12.sky.zip2020-11-21 16:35:58115163331
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