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
Download 19:58:15101362386,13112 20:32:511,48717,12017,12085,5765 20:43:065264022029 11:18:18818116286694 10:51:36818116285693 10:48:30166625 20:07:55214147521 11:44:41818116291496 13:59:10131402166
Pilot Kinetics Experiment CVN Mice 12:35:161012188
Pilot Experiment APOE3 3 Mice (21 DBS_3Plasma_2Brain) 12:29:3410162226
E4HN 69 Wk 12:27:5910172384 08:41:122,78415,73615,736112,55724 08:41:113,42618,79218,792130,95824 22:30:1090961921,14696 22:00:20909619299684 21:58:5590961921,58694 21:34:3590961921,53696 21:10:3590961921,15896 20:45:4290961921,53896 20:22:5990961922,75496 19:57:1690961921,24296 19:28:3390961922,76496 18:45:44909619298295 18:44:3190961922,76494 18:08:05909619298696 18:06:41909619299095 18:04:43909619297895 18:02:4890961922,76494 17:37:47909619283495 17:36:23909619275296 17:35:27909619279696 17:34:11909619278596 17:32:2190961921,06496 16:42:31909619273296 16:41:36909619296096 16:40:3390961921,09695 16:02:0390961921,07496 15:42:0390961921,16096 15:22:0390961921,05495 15:01:3290961921,12295 13:42:2190961921,20296 12:29:44818116284896 12:28:15818116282496 12:26:30818116283289 12:24:58818116288296 12:23:20818116286696 12:21:57818116276996 12:20:32818116287696 12:19:10818116279095 12:17:40818116286896 12:16:10606012051896 12:15:29606012050496 12:14:32606012051696 12:13:39606012051896 12:12:51606012050696 12:12:12606012051695 12:11:32606012050696 12:10:47606012049296 12:09:55616112252896 12:08:50595911848493 12:07:52595911850296 12:06:52606012050693 12:05:51606012051496 12:04:35606012052296 12:03:50606012050696 12:02:40606012051696 12:01:46595911851696 12:00:02606012053296 08:02:333310310347437 15:24:0010282816220 15:24:005131312013
EGFR pathway peptide standards at different DDM concentrations 19:49:41101010325 02:16:567292,1523,03921,74141 02:16:564,08520,25840,417130,13220 02:16:566,32343,96187,681564,73484 02:16:566,32343,96187,681301,78784 17:01:46111123489 17:01:40720376528 17:01:35111123327 17:01:32113143940 17:01:32113133712 17:01:29110103030 17:01:26110103050 17:01:23117228500 17:00:32112732883,37720 16:52:03719356210 16:51:367203766105 16:51:3672037111100 16:49:38112732883,37727 16:49:38112732883,37727 16:36:23113132620 16:36:23113132621 16:36:23113132621 16:36:23113132620 16:35:581661910 16:35:58616173810 16:35:58113142611 16:35:58113164911 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
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