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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DDRpanel2_LCL_time_2021-07-16_11-50-45.sky.zip2021-07-19 08:33:26325911884730
DDRpanel2_PBMC_2021-07-16_11-50-34.sky.zip2021-07-19 08:33:26325911884125
DDRpanel1_PBMC_2021-07-16_11-50-24.sky.zip2021-07-19 08:33:26377014054226
DDRpanel2_LCL_dose_2021-07-16_11-50-14.sky.zip2021-07-19 08:33:26325911884715
20180731_UPR_ARoos_H&L_paper_Lumos_RT.sky.zip2021-07-16 07:57:138214214228
Nrxn3 HA IP splice segment analysis2021-07-13 08:24:0947147011
Nrxn3 AS5 knock-out analysis2021-07-13 08:17:391153216631
Nrxn3 Exon 25 Titration Curve2021-07-13 08:15:0518169214
Figure 5.sky.zip2021-07-10 09:08:069203410230
Figure 4.sky.zip2021-07-10 09:08:069203410222
Figure 3D.sky.zip2021-07-10 09:08:059203410227
Figure 3C.sky.zip2021-07-10 09:08:059203410227
Figure 3B.sky.zip2021-07-10 09:08:059203410222
Figure 3A.sky.zip2021-07-10 09:08:059203410222
PRM Splice Assays2021-06-24 08:36:0739841659900
DDR2_response curve_2021-06-17_11-52-03.sky.zip2021-06-24 08:35:5527469264434
210429to210501_ApoE_5x5_Precision.sky2021-06-22 12:52:48151024139
DDR2_chromatogram8_2021-06-21_13-22-59.sky.zip2021-06-22 08:22:184510801
DDR2_chromatogram8_2021-06-21_13-22-59.sky.zip2021-06-21 13:23:364510801
DDR2_response curve_2021-06-17_11-52-03.sky.zip2021-06-17 11:52:4827469264434
Bruderer.sky.zip2021-06-11 09:20:413,86625,31129,238175,3950
Selevsek.sky.zip2021-06-08 13:46:483,59738,88950,764304,5840
BSA-QC_68fmol_Apr2021_Impact-II_2021-05-20_15-45-54.sky.zip2021-05-20 15:46:21131402164
210512_Trypsin_P100_upload_2021-05-12_13-19-24.sky.zip2021-05-12 04:19:42902765792,8570
210512_Lysarginase_P100_upload_2021-05-12_13-17-23.sky.zip2021-05-12 04:17:39741633411,6630
20200704_ITvOT_plasma_final_2021-04-28_13-32-15.sky.zip2021-04-28 20:58:381544344342,98414
qLITvsqOT_matrix_matched_originaltransitions_LIT_2021-04-27_14-24-59.sky.zip2021-04-28 20:58:381244234232,95933
qLITvsqOT_matrix_matched_originaltransitions_OT_2021-04-27_14-23-49.sky.zip2021-04-28 20:58:381244234232,95933
qLITvsqOT_matrix_matched_alltransitions_OT_2021-04-27_14-18-08.sky.zip2021-04-28 20:58:381244234236,20233
qLITvsqOT_matrix_matched_alltransitions_LIT_2021-04-27_14-13-12.sky.zip2021-04-28 20:58:381244234236,20233
qLITvsqOT_precision_final_2020-12-30_07-35-14.sky.zip2021-04-28 20:58:381284604603,01420
LINCS_GCP_Plate70_annotated_minimized_2019-12-16_17-22-40.sky.zip2021-04-21 20:21:56818116280285
LINCS_GCP_Plate94_annotated_minimized_2020-04-03_16-19-23.sky.zip2021-04-18 11:14:24818116285096
BSA-QC_68fmol_Jan-Feb2021_Impact-II_2021-04-16_07-04-15.sky.zip2021-04-16 07:04:33131402164
MRM Brain_2021-02-24_11-23-27.sky.zip2021-04-13 15:28:2822459026841
MRM Heart_2021-02-26_13-08-25.sky.zip2021-04-13 15:28:2822438625638
MRM Cerebelum_2021-02-24_13-23-28.sky.zip2021-04-13 15:28:2822438625640
DDR2_chromatogram5.sky.zip2021-04-08 06:02:52112141
DDR2_chromatogram1.sky.zip2021-04-07 18:04:172738765261
DDR2_chromatogram2.sky.zip2021-04-07 18:03:1078161101
DDR2_chromatogram4.sky.zip2021-04-07 18:02:2011281
DDR2_chromatogram3.sky.zip2021-04-07 18:01:31112141
ARP_Plasma_0.2_vs_2.0_mg_2021-02-24_18-55-48.sky.zip2021-04-02 14:46:32404124891,40511
ARP-OxHSA-PlasmaMatrix_DilSeries_2021-02-25_11-31-34.sky.zip2021-04-02 14:46:32224129686218
Plasma-OxHSA_2021-02-25_11-37-12.sky.zip2021-04-02 14:46:32626330991811
3cm_preliminary_data_2021-03-26_18-17-25.sky.zip2021-03-31 10:24:111371312
20190927 PIP extraction efficiency.sky.zip2021-03-29 06:45:2710114122
20190928 Donor 6-7 PIPs MRM 190926.sky.zip2021-03-29 06:45:2610114130
20190929 Extraction efficiency.sky.zip2021-03-29 06:45:2610114120
20191002 Brain samples.sky.zip2021-03-29 06:45:2610114113
20191003 Donor 6-7 + calibration curve.sky.zip2021-03-29 06:45:2610114136
20191105 Calibration curve.sky.zip2021-03-29 06:45:2610114130
20191108 Gradient.sky.zip2021-03-29 06:45:261011413
D2 15 CRP.sky.zip2021-03-29 06:45:2610114120
20191114 Extraction efficiency.sky.zip2021-03-29 06:45:2610114119
PIPs MRM 191001 Donor 45.sky.zip2021-03-29 06:45:2610114124
PIPs MRM 190930 OP9.sky.zip2021-03-29 06:45:2610114111
LINCS_P100_DIA_Plate15_annotated_minimized_2018-05-02_19-32-22.sky.zip2021-03-19 20:17:4890961921,20296
LINCS_P100_DIA_Plate16_annotated_minimized_2018-05-02_19-43-10.sky.zip2021-03-19 20:17:4890961921,12295
LINCS_P100_DIA_Plate17_annotated_minimized_2018-05-02_17-35-29.sky.zip2021-03-19 20:17:4890961921,05495
LINCS_P100_DIA_Plate19_annotated_minimized_2018-05-02_17-18-22.sky.zip2021-03-19 20:17:4890961921,16096
LINCS_P100_DIA_Plate20_annotated_minimized_2018-05-02_21-04-33.sky.zip2021-03-19 20:17:4890961921,07496
LINCS_P100_DIA_Plate22_annotated_minimized_2018-05-02_22-33-32.sky.zip2021-03-19 20:17:4890961921,09695
LINCS_P100_DIA_Plate23_annotated_minimized_2018-05-02_18-13-55.sky.zip2021-03-19 20:17:48909619296096
LINCS_P100_DIA_Plate24_annotated_minimized_2018-05-02_19-07-44.sky.zip2021-03-19 20:17:48909619273296
LINCS_P100_DIA_Plate27_annotated_minimized_2018-05-02_19-16-47.sky.zip2021-03-19 20:17:4890961921,06496
LINCS_P100_DIA_Plate30_annotated_minimized_2018-05-02_18-33-32.sky.zip2021-03-19 20:17:48909619278596
LINCS_P100_DIA_Plate33_annotated_minimized_2018-05-02_17-47-07.sky.zip2021-03-19 20:17:48909619279696
LINCS_P100_DIA_Plate34_annotated_minimized_2019-08-30_17-25-21.sky.zip2021-03-19 20:17:48909619275296
LINCS_P100_DIA_Plate58_annotated_minimized_2017-11-28_11-50-08.sky.zip2021-03-19 20:17:48909619283495
LINCS_P100_DIA_Plate59a_annotated_minimized_2018-11-30_14-13-58.sky.zip2021-03-19 20:17:4890961922,76494
LINCS_P100_DIA_Plate60_annotated_minimized_2017-11-29_16-22-26.sky.zip2021-03-19 20:17:48909619297895
LINCS_P100_DIA_Plate61_annotated_minimized_2018-02-06_17-09-06.sky.zip2021-03-19 20:17:48909619299095
LINCS_P100_DIA_Plate62_annotated_minimized_2018-04-20_15-55-05.sky.zip2021-03-19 20:17:48909619298696
LINCS_P100_DIA_Plate63a_annotated_minimized_2019-11-14_15-28-26.sky.zip2021-03-19 20:17:4890961922,76494
LINCS_P100_DIA_Plate64_annotated_minimized_2018-05-02_20-34-19.sky.zip2021-03-19 20:17:48909619298295
LINCS_P100_DIA_Plate66_annotated_minimized_2018-08-31_10-36-13.sky.zip2021-03-19 20:17:4790961922,76496
LINCS_P100_PRM_Plate25_annotated_minimized_2018-05-02_18-24-10.sky.zip2021-03-19 20:17:4790961921,24296
LINCS_P100_DIA_Plate67_annotated_minimized_2018-08-02_13-56-24.sky.zip2021-03-19 20:17:4790961922,75496
LINCS_P100_PRM_Plate18_annotated_minimized_2020-01-08_17-11-19.sky.zip2021-03-19 20:17:4790961921,53896
LINCS_P100_PRM_Plate28_annotated_minimized_2018-05-02_18-51-02.sky.zip2021-03-19 20:17:4790961921,15896
LINCS_P100_PRM_Plate29_03H_annotated_minimized_2018-05-02_21-16-28.sky.zip2021-03-19 20:17:4790961921,53696
LINCS_P100_DIA_Plate65_annotated_minimized_2019-10-21_16-54-32.sky.zip2021-03-19 20:17:4790961921,58694
LINCS_P100_PRM_Plate31_annotated_minimized_2018-05-02_18-00-17.sky.zip2021-03-19 20:17:47909619299684
LINCS_P100_PRM_Plate32_annotated_minimized_2018-05-02_20-45-26.sky.zip2021-03-19 20:17:4790961921,14696
ZaharaN_MM_Final_SkylineReport_2020-07-30_12-54-33.sky.zip2021-03-18 16:36:239212321832
Martinez et al_PRM Replicates 5-7_MCP_2021-01-28_10-13-34.sky.zip2021-03-18 16:36:23919192378
DDR2_repeatability2.sky.zip2021-03-18 07:29:152365057
DDR2_repeatability1.sky.zip2021-03-18 07:29:0931499868857
DDR2_response curve2.sky.zip2021-03-18 07:22:231242827
Diab_tear_SRM_2021-03-18_13-32-33.sky.zip2021-03-18 05:33:45182649102112
BSA_method_dev.sky.zip2021-03-14 13:29:58143545443
BSA_and_PRTC.sky.zip2021-03-14 13:29:5361021171,1683
Meningioma_CSF_MRM_2021-02-17_13-51-29.sky.zip2021-03-11 11:32:2851515627
Glioma_CSF_MRM_2021-02-17_13-57-43.sky.zip2021-03-11 11:32:28515156410
Medulloblastoma_MRM_2021-02-17_14-29-27.sky.zip2021-03-11 11:32:2831010539
MCF-7_QC_Analysed_2021-02-17_14-54-14.sky.zip2021-03-11 11:32:28266675
BSA_QC_Analysed_2021-02-17_14-56-13.sky.zip2021-03-11 11:32:28177655
BSA_QC_standard curve_2021-02-17_14-57-03.sky.zip2021-03-11 11:32:28177655
Glioma_tissue_MRM_2021-03-11_17-52-52.sky.zip2021-03-11 11:32:28312121039
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