Schilling - MRM_HR_TripleTOF_MS2_FullScanFiltering

Schilling - MRM_HR_TripleTOF_MS2_FullScanFiltering
Multiplexed, scheduled high resolution (sMRM-HR) acquisition on a full scan QqTOF instrument with integrated data-dependent and targeted mass spectrometric workflows.
  • Organism: Bos taurus, Saccharomyces cerevisiae, Escherichia 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

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
 
Download
TPAD_CSF1A_Batch1-multiNF-DIANN.sky.zip2025-07-01 17:49:472,85327,12832,575259,53243
TPAD_CSF1A_Batch1-multiNF-DIANN-grouped.sky.zip2025-07-01 17:41:312,67226,43931,769253,11943
TPAD_CSF1B_Batch2-multiNF-DIANN.sky.zip2025-07-01 15:26:132,85327,12832,575259,53241
TPAD_CSF1B_Batch2-multiNF-DIANN-grouped.sky.zip2025-07-01 15:17:422,67226,43931,769253,11941
TPAD_CSF2A_Batch3-multiNF-DIANN.sky.zip2025-07-01 14:13:302,85327,12832,575259,53243
TPAD_CSF2A_Batch3-multiNF-DIANN-grouped.sky.zip2025-07-01 14:04:582,67226,43931,769253,11943
TPAD_CSF2B_Batch4-multiNF-DIANN.sky.zip2025-07-01 11:21:372,85327,12832,575259,53243
TPAD_CSF2B_Batch4-multiNF-DIANN-grouped.sky.zip2025-07-01 11:09:482,67226,43931,769253,11943
TPAD_CSF3A_Batch5-multiNF-DIANN.sky.zip2025-06-30 15:31:332,85327,12832,575259,53243
TPAD_CSF3A_Batch5-multiNF-DIANN-grouped.sky.zip2025-06-30 15:23:252,67226,43931,769253,11943
TPAD_CSF3B_Batch6-multiNF-DIANN.sky.zip2025-06-30 14:43:542,85327,12832,575259,53241
TPAD_CSF3B_Batch6-multiNF-DIANN-grouped.sky.zip2025-06-30 14:36:142,67226,43931,769253,11941
TPAD_CSF4_Batch7-multiNF-DIANN.sky.zip2025-06-30 13:55:172,85327,12832,575259,53242
TPAD_CSF4_Batch7-multiNF-DIANN-grouped.sky.zip2025-06-30 13:46:482,67226,43931,769253,11942
TPAD_CSF5_Batch8-multiNF-DIANN.sky.zip2025-06-30 13:02:062,85327,12832,575259,53254
TPAD_CSF5_Batch8-multiNF-DIANN-grouped.sky.zip2025-06-30 11:52:172,67226,43931,769253,11954
METRIC_Eclipse_PSB_All_QCs_2025-06-27_01-27-04.sky.zip2025-06-26 22:27:151666233
Exp_CSF_GPF_A_2024-05-06_21-16-47.sky.zip2025-06-25 18:12:201,8297,3787,37855,0401
2024-05 CSF LIT GPF-MSFragger-3.sky.zip2025-06-25 18:12:201,0825,0956,01940,4016
240207_gpf_2024-06-04_10-24-18.sky.zip2025-06-25 18:12:201,2886,98410,593126,0861
230305_ev2_prm_final_inj1_2024-06-04_07-32-10.sky.zip2025-06-25 18:12:207222,0892,08914,44942
230124_p2_neo_30min_3500targets_opt_trans_pepleveldilution_2024-06-04_06-58-16.sky.zip2025-06-25 18:12:201,0273,5013,50111,42041
CSF_neurod105_assay_individuals_manual_2024-06-03_15-26-36.sky.zip2025-06-25 18:12:201029029026,34537
Exp_CSF_MMCC_quant_all_adjBound_opttrans_nochick_2024-06-03_14-53-02.sky.zip2025-06-25 18:12:201,2058,3748,37424,93427
OT_GPF_PRM_survey_MMCC_boundaries_opttrans_nochick_2024-06-03_14-36-08.sky.zip2025-06-25 18:12:201,2801,9711,9715,80127
LIT_GPF_survey_newAlign_MMCC_boundaries_opttrans_nochick_2024-06-02_15-26-11.sky.zip2025-06-25 18:12:207982,0352,0355,99827
Aducanumab_Selectivity_2025-06-19_12-58-48.sky.zip2025-06-19 09:59:0644851236
hela_isolation_window_concentration_2000ng.sky.zip2025-06-02 18:36:1910,365188,947223,7653,101,14818
hela_isolation_window_experiment_200ng.sky.zip2025-06-02 18:36:1910,365188,947223,7653,101,14818
hela_isolation_window_experiment_500ng.sky.zip2025-06-02 18:36:1910,364188,947223,7653,101,14818
hela_isolation_window_experiment_1000ng.sky.zip2025-06-02 18:36:1910,365188,947223,7653,101,14818
2025-0405-MMCC-EV-Astral-noimpute_2025-05-29_11-52-05.sky.zip2025-06-02 18:36:194,90737,54742,331335,89142
2025-0405-MMCC-EV-Actis-noimpute_2025-05-29_11-46-45.sky.zip2025-06-02 18:36:195,18541,97847,810380,07242
SMTG-low-value-imputed_2025-05-01_14-22-15.sky.zip2025-06-01 21:38:1710,383187,220217,1092,214,24878
SMTG-MissForest-imputed_2025-05-01_11-41-41.sky.zip2025-06-01 20:04:5710,383187,220217,1092,214,24878
SMTG-nettle-imputed_2025-04-30_12-25-18.sky.zip2025-06-01 18:16:1410,639215,139251,4792,483,95978
SMTG-unimputed_2025-04-30_08-55-40.sky.zip2025-06-01 13:32:5610,639215,139251,4792,483,95978
TEI-REX-unimputed_2025-04-29_15-46-15.sky.zip2025-06-01 11:49:381,30749,85259,007585,98796
TEI-REX-nettle-imputed_2025-04-29_13-36-50.sky.zip2025-06-01 11:15:251,30749,85259,007585,98796
MagNet-holdout-nettle-imputed_2025-05-01_08-50-25.sky.zip2025-06-01 09:40:175,29914,11015,07460,28842
hela_isolation_window_concentration_2000ng.sky.zip2025-05-30 15:25:4610,365188,947223,7653,101,14818
ecoli_large_replicates_loaded_refined_2024-05-30_14-02-04.sky.zip2025-05-30 12:00:183669899894,9408
ecoli_large_replicates_loaded_2024-05-30_14-00-59.sky.zip2025-05-30 12:00:184611,3021,3027,5248
ecoli_large_replicates_2024-05-30_14-00-05.sky.zip2025-05-30 12:00:184611,3021,3027,5240
ecoli_subset_replicates_refined_cv_2024-05-30_13-58-18.sky.zip2025-05-30 12:00:187592,2662,26612,8072
ecoli_subset_replicates_refined_2024-05-30_13-57-25.sky.zip2025-05-30 12:00:187592,4372,43713,6022
ecoli_subset_replicates_2024-05-30_13-56-16.sky.zip2025-05-30 12:00:187592,4372,44130,8572
gpf_results_importer_2024-05-30_13-54-14.sky.zip2025-05-30 12:00:181,1925,3967,42785,6731
gpf_results_manual_2024-05-30_13-52-23.sky.zip2025-05-30 12:00:181,1925,3967,42785,6731
pq500_100spd_plasma_final_lightheavy_replicates_2024-05-30_13-50-10.sky.zip2025-05-30 11:58:415798181,62213,69910
pq500_60spd_plasma_final_lightheavy_replicates_2024-05-30_13-48-26.sky.zip2025-05-30 11:58:415798181,62213,87610
pq500_100spd_plasma_final_replicates_2024-05-30_13-47-38.sky.zip2025-05-30 11:58:415798188187,08110
pq500_60spd_plasma_final_replicates_2024-05-30_13-46-43.sky.zip2025-05-30 11:58:415798188186,97710
pq500_100spd_plasma_multireplicate_results_refined_2024-05-30_13-46-07.sky.zip2025-05-30 11:58:415798188187,0811
pq500_60spd_plasma_multireplicate_results_refined_2024-05-30_13-45-40.sky.zip2025-05-30 11:58:415798188186,9771
pq500_100spd_plasma_multireplicate_results_2024-05-30_13-45-07.sky.zip2025-05-30 11:58:415798188187,6512
pq500_60spd_plasma_multireplicate_results_2024-05-30_13-44-21.sky.zip2025-05-30 11:58:415798188187,6012
pq500_100spd_neat_multireplicate_results_refined_2024-05-30_13-43-42.sky.zip2025-05-30 11:58:415798188187,6891
pq500_60spd_neat_multireplicate_results_refined_2024-05-30_13-43-05.sky.zip2025-05-30 11:58:415798188187,6011
pq500_100spd_neat_multireplicate_results_2024-05-30_13-42-26.sky.zip2025-05-30 11:58:4157981881811,0881
pq500_60spd_neat_multireplicate_results_2024-05-30_13-41-26.sky.zip2025-05-30 11:58:4157981881811,0881
pq500_60spd_neat_multireplicate_2024-05-30_13-40-53.sky.zip2025-05-30 11:58:4157981881811,0880
hela_isolation_window_experiment_200ng.sky.zip2025-05-30 01:29:3910,365188,947223,7653,101,14818
hela_isolation_window_experiment_500ng.sky.zip2025-05-30 00:53:5310,364188,947223,7653,101,14818
hela_isolation_window_experiment_1000ng.sky.zip2025-05-29 23:15:1110,365188,947223,7653,101,14818
2025-0405-MMCC-EV-Astral-noimpute_2025-05-29_11-52-05.sky.zip2025-05-29 12:13:324,90737,54742,331335,89142
2025-0405-MMCC-EV-Actis-noimpute_2025-05-29_11-46-45.sky.zip2025-05-29 12:03:295,18541,97847,810380,07242
20240709_OBIPhA.sky.zip2025-05-16 09:25:513333723512
Swaney-IPMS-Data-DIANN_2025-05-15_11-53-43.sky.zip2025-05-15 12:02:265,18545,78152,836422,66115
Vial Comparison Post 01152025 v1 (not daily)_2025-04-29_15-52-29.sky.zip2025-04-30 21:22:2310242419
Vial Comparison Pre v2_2025-04-29_15-49-25.sky.zip2025-04-30 21:22:2310242423
ATI_Samples_2023_2025-04-26_13-39-03.sky.zip2025-04-28 10:30:1413244828042
ATI_Response_2023_2025-04-26_13-25-06.sky.zip2025-04-28 10:30:1413244828018
ATI_Samples_2022_2025-02-20_23-31-07.sky.zip2025-04-28 10:30:1012234626848
ATI_Samples_2021_2025-02-20_23-21-52.sky.zip2025-04-28 10:30:0812234626830
2023_ATIsamples_circe_2025-02-20_13-24-11.sky.zip2025-04-25 14:58:1713224325096
2022_ATIsamples_circe_2025-02-20_13-17-06.sky.zip2025-04-25 14:58:0912224325097
2021_ATIsamples_circe_2025-02-20_13-06-53.sky.zip2025-04-25 14:58:0212224325094
SICK_glyco_DIA_OrbitrapSCE_library_2025-04-19_01-42-54.sky.zip2025-04-19 17:49:212455,7962,69935,937100
SICK_glyco_DIA_AstralCE_library_2025-04-19_01-01-56.sky.zip2025-04-19 17:49:212568,9083,80054,729100
051024_PlasmaPilotwithDose_2025-04-10_16-09-16.sky.zip2025-04-10 13:09:4114848366
050124_CortexMatrixSelectivityTesting_2025-04-10_15-56-24.sky.zip2025-04-10 12:56:481485260
METRIC_Exploris_PSB_All_QCs_2025-04-10_15-22-54.sky.zip2025-04-10 12:22:09166629
Histonas_654187687vez_2025-04-10_14-40-28.sky.zip2025-04-10 05:47:5400000
MS3_15N_dilution_2025-04-04_14-57-13.sky.zip2025-04-07 04:48:0677113776051
Transition_1_Astral_Stellar_second_set_cut_off.sky.zip2025-04-07 04:48:069013,1103,57514,81722
Transition_1_Astral_Stellar_second_set.sky.zip2025-04-07 04:48:069014,7315,43722,64622
pdDiscoverer_GPF.sky.zip2025-04-07 04:48:063962,4523,54842,2186
Intermediate_1_Astral_Stellar_second_subset_CV30.sky.zip2025-04-07 04:48:064811,9972,22411,4778
Intermediate_1_Astral_Stellar_second_subset.sky.zip2025-04-07 04:48:065532,5002,82314,4028
GPF_plasma_refined_210prot.sky.zip2025-04-07 04:48:062106847394,5003
GPF_plasma_intermediate.sky.zip2025-04-07 04:48:063221,5701,90712,7529
GPF_plasma.sky.zip2025-04-07 04:48:063221,5701,90712,7529
final_assay_Astral_Stellar_410.sky.zip2025-04-07 04:48:064091,2361,3056,2443
all15N_peptides_absolute_quantification.sky.zip2025-04-07 04:48:067561124983
15N_dilution.sky.zip2025-04-07 04:48:0675711465452
Unknown-HMW-GS-Combination_2024-12-12_13-43-29.sky.zip2025-04-01 14:47:3114404039258
Known-HMW-GS-Combination_2024-12-12_13-15-23.sky.zip2025-04-01 14:47:2614404039237
07_Final-Method_Pooled-Sample_2024-12-12_13-01-40.sky.zip2025-04-01 14:47:211440403814
06_Refinement-Method_Pooled-Sample_2024-12-12_13-01-05.sky.zip2025-04-01 14:47:211467671,0164