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 (834 KB)2019-07-1515101006 (814 KB)2019-07-1515101006 (843 KB)2019-07-1515101006 (834 KB)2019-07-15158806 (2 MB)2019-07-151144024 (2 MB)2019-07-151267834 (1 MB)2019-07-0811254516814 (431 KB)2019-07-081125451683 (2 MB)2019-07-0811254516820 (1 MB)2019-07-0811254516814 (1 MB)2019-07-0811254516814 (2 MB)2019-07-0811254516823 (2 MB)2019-07-0811254516823 (2 MB)2019-07-0811254516824 (2 MB)2019-07-0811254516820 (21 MB)2019-07-081125451686 (3 MB)2019-07-0820294918224 (2 MB)2019-07-0820294918222 (2 MB)2019-07-0820294918218 (2 MB)2019-07-0820294918223 (3 MB)2019-07-0820294918230 (2 MB)2019-07-0820294918220 (2 MB)2019-07-0820294918223 (12 MB)2019-06-281062752751,07116 (5 MB)2019-06-2820878734118 (33 MB)2019-06-281906636632,63520 (12 MB)2019-06-28972812811,08316 (1 MB)2019-06-2710204015414 (1 MB)2019-06-2711204015620 (1 MB)2019-06-2710204015415 (1 MB)2019-06-2711204015615 (121 MB)2019-06-1390961922,76496 (12 MB)2019-06-10818116283018 (5 MB)2019-06-1081811628548 (81 MB)2019-06-1090961922,76266 (140 MB)2019-06-0890961922,76496 (110 MB)2019-06-0890961922,76491 (100 MB)2019-06-0790961922,75866 (7 MB)2019-06-04882417496 (8 MB)2019-06-04882418396 (7 MB)2019-06-03882316196 (140 MB)2019-06-0390961922,76496 (7 MB)2019-06-03882416596
all (46 MB)2019-05-31133575268,5337
20180518 multiplex quantification - APOA4 only (265 MB)2019-05-311364864
20180518 multiplex quantification only (380 MB)2019-05-3128357050268 (120 MB)2019-05-311613628684820
500 µE (PG-500uE_2ul_2019-05-16_15-23-09) (7 MB)2019-05-292710510555811 (3 MB)2019-05-2920611225758 (13 MB)2019-05-2931681366528
200µE (R5-8_V2_2019-05-16_15-31-29) (8 MB)2019-05-2928979751616 (1 GB)2019-05-283,22827,28927,28985,17542 (1 GB)2019-05-243,73136,39336,39395,63942 (162 MB)2019-05-2220989862412 (531 MB)2019-05-224,00716,22916,22947,85630 (1 GB)2019-05-223,48230,60230,60290,48842
Janschitz et al_PRM_results_Panorama (611 MB)2019-05-094475833712 (16 MB)2019-04-29818116287833 (43 MB)2019-04-26909619292496 (128 MB)2019-04-2690961921,00296 (52 MB)2019-04-18909619278892 (6 MB)2019-04-18882413292 (19 MB)2019-04-17818116291233 (26 MB)2019-04-16818116291233 (24 MB)2019-04-16808016080533 (24 MB)2019-04-16818116289433 (4 MB)2019-04-08882415666 (72 MB)2019-04-0890961922,76466 (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 (1 MB)2019-04-062221318 (75 MB)2019-04-0290961922,76466 (2 MB)2019-04-01882417436 (39 MB)2019-04-0190961922,76236 (4 MB)2019-04-0172020935 (5 MB)2019-03-28882416266 (6 MB)2019-03-27882417182 (98 MB)2019-03-2790961922,76284 (148 MB)2019-03-265353514748 (7 MB)2019-03-13882416596
HLF with (176 KB)2019-03-086045451
U87MG with (429 KB)2019-03-08901181181
IgA with (358 KB)2019-03-08801251251
HNE with (174 KB)2019-03-086038381
PGM OG with (370 KB)2019-03-08201061061
IgG with (228 KB)2019-03-087061611
Fet NG with (229 KB)2019-03-087063631
CBHI OG with (93 KB)2019-03-082020201
CBHI NG with (109 KB)2019-03-083028281
BLF with (302 KB)2019-03-088085851 (429 KB)2019-03-08901191191 (40 KB)2019-03-08103181 (797 KB)2019-03-0810151510 (63 MB)2019-03-088011634212 (6 MB)2019-03-0820183216
PRM-Identifying the N-terminal amino acid for a TAG start (3 MB)2019-03-05266583
PRM-Identifying the N-terminal amino acid for an ATG start (1 MB)2019-03-05266563 (5 MB)2019-03-01551332582,2633 (5 MB)2019-03-01631342602,0613 (51 MB)2019-02-2674730,02544,411576,8660 (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
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