Schilling - MS1_MS2_SWATH

Schilling - MS1_MS2_SWATH

MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments.

 

Authors: Matthew J. Rardin, Birgit Schilling, Lin-Yang Cheng, Brendan X. MacLean, Dylan J. Sorensen, Alexandria K. Sahu, Michael J. MacCoss, Olga Vitek, and Bradford W. Gibson.

 

Data sets acquired on a TripleTOF 5600 mass spectrometer and uploaded to Panorama:

 

  • MS1 and SWATH-MS – Kinase Inhibitor Study for quantifying changes in endogenous phosphopeptide isomers YHGHpS293MSDPGVSYR and YHGHSMSDPGVpS300YR from PDHE1a (3 injection replicates), go to Kinase Inhibitor Study in Panorama, download Kinase Inhibitor Study.
  • MS1 and SWATH-MS – dilution curve of heavy labeled phosphopeptide isomers YHGHpS293MSDPGVSYR and YHGHSMSDPGVpS300YR from PDHE1a (3 injection replicates), download various isomer ratio sub files: 16:1/1:168:1/1:84:1/1:4, 1:2, 1:1.

 

 

  • MS1 and SWATH-MS – MS1 and MS2 Data Processing Study for removal of interferences using mitochondrial lysates at a concentration of 100 ng or 33 ng (2 process replicates, 3 injection replicates), go to Data Processing Study in Panorama, download Data Processing Study (sample legend).

Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
tfreedman_brian023_20190605_17363_LMWstim_2019-07-11_11-35-04.sky.zip (834 KB)2019-07-1515101006
tfreedman_brian023_20190605_17363_LMWnostim_2019-07-11_11-34-30.sky.zip (814 KB)2019-07-1515101006
tfreedman_brian023_20190605_17363_HMWstim_2019-07-11_11-34-02.sky.zip (843 KB)2019-07-1515101006
tfreedman_brian023_20190605_17363_HMWnostim_2019-07-11_11-33-22.sky.zip (834 KB)2019-07-15158806
tfreedman_brian023_20190605_17363_Calibration_2019-07-09_12-03-07.sky.zip (2 MB)2019-07-151144024
calcurve_pY32_unmodifiedY32_2019-07-09_11-44-05.sky.zip (2 MB)2019-07-151267834
Round2_S26_110515_Controls5_2019-07-08_13-48-30.sky.zip (1 MB)2019-07-0811254516814
Round2_S26_missing_Controls10_2019-07-08_13-47-57.sky.zip (431 KB)2019-07-081125451683
Round2_S26_113015_Controls9_2019-07-08_13-47-03.sky.zip (2 MB)2019-07-0811254516820
Round2_S26_111315_Controls8_2019-07-08_13-45-55.sky.zip (1 MB)2019-07-0811254516814
Round2_S26_111115_Controls7_2019-07-08_13-45-14.sky.zip (1 MB)2019-07-0811254516814
Round2_S26_110715_Controls6_2019-07-08_13-44-11.sky.zip (2 MB)2019-07-0811254516823
Round2_S26_110315_Controls4_2019-07-08_13-43-07.sky.zip (2 MB)2019-07-0811254516823
Round2_S26_110215_Controls3_2019-07-08_13-42-11.sky.zip (2 MB)2019-07-0811254516824
Round2_S26_101815_Controls2_2019-07-08_13-39-51.sky.zip (2 MB)2019-07-0811254516820
Round2_S26_033116_missing_Controls1_2019-07-08_13-38-37.sky.zip (21 MB)2019-07-081125451686
032115_S26_Results_Controls7_2019-07-08_13-30-46.sky.zip (3 MB)2019-07-0820294918224
032015_S26_Results_Controls6_2019-07-08_13-30-12.sky.zip (2 MB)2019-07-0820294918222
031715_S26_Results_Controls5_2019-07-08_13-29-40.sky.zip (2 MB)2019-07-0820294918218
031615_S26_Results_Controls4_2019-07-08_13-28-56.sky.zip (2 MB)2019-07-0820294918223
031315_S26_Results_Controls3_2019-07-08_13-28-13.sky.zip (3 MB)2019-07-0820294918230
031215_S26_Results_Controls2_2019-07-08_13-27-37.sky.zip (2 MB)2019-07-0820294918220
031115_S26_Results_Controls1_2019-07-08_13-26-51.sky.zip (2 MB)2019-07-0820294918223
CUL5_HIV_interactors_quant_2018-02-16_10-51-21.sky.zip (12 MB)2019-06-281062752751,07116
CBFB_HIV_CUL2_CUL5_interactors_quant_2018-02-16_10-59-32.sky.zip (5 MB)2019-06-2820878734118
ELOB_HIV_interactors_quant_2018-02-16_11-00-53.sky.zip (33 MB)2019-06-281906636632,63520
CBFB_HIV_interactors_quant_2018-02-16_10-55-27.sky.zip (12 MB)2019-06-28972812811,08316
Healthy_controls_round2_042617_2019-06-27_16-10-07.sky.zip (1 MB)2019-06-2710204015414
Healthy_controls_round1_042617_2019-06-27_16-09-16.sky.zip (1 MB)2019-06-2711204015620
103015_LewinsohnNegatives_round2_2019-06-27_16-02-00.sky.zip (1 MB)2019-06-2710204015415
103015_LewinsohnNegatives_round1_2019-06-27_15-31-23.sky.zip (1 MB)2019-06-2711204015615
LINCS_P100_DIA_Plate80_annotated_minimized_2019-06-13_12-13-28.sky.zip (121 MB)2019-06-1390961922,76496
LINCS_GCP_Plate71b_annotated_minimized_2019-06-10_18-12-11.sky.zip (12 MB)2019-06-10818116283018
LINCS_GCP_Plate71a_annotated_minimized_2019-06-10_18-10-31.sky.zip (5 MB)2019-06-1081811628548
LINCS_P100_DIA_Plate77_annotated_minimized_2019-06-10_16-17-36.sky.zip (81 MB)2019-06-1090961922,76266
LINCS_P100_DIA_Plate70_annotated_minimized_2019-06-03_13-47-33.sky.zip (140 MB)2019-06-0890961922,76496
LINCS_P100_DIA_Plate82_annotated_minimized_2019-06-03_16-03-24.sky.zip (110 MB)2019-06-0890961922,76491
LINCS_P100_DIA_Plate76_annotated_minimized_2019-06-07_15-29-16.sky.zip (100 MB)2019-06-0790961922,75866
LINCS_P100_QC_Plate80_annotated_minimized_2019-06-04_16-27-57.sky.zip (7 MB)2019-06-04882417496
LINCS_P100_QC_Plate82_annotated_minimized_2019-06-04_15-46-59.sky.zip (8 MB)2019-06-04882418396
LINCS_P100_QC_Plate78_annotated_minimized_2019-06-03_15-57-25.sky.zip (7 MB)2019-06-03882316196
LINCS_P100_DIA_Plate70_annotated_minimized_2019-06-03_13-47-33.sky.zip (140 MB)2019-06-0390961922,76496
LINCS_P100_QC_Plate70_annotated_minimized_2019-06-03_13-48-35.sky.zip (7 MB)2019-06-03882416596
all combined_2019-03-21_20-40-14.sky.zip (46 MB)2019-05-31133575268,5337
20180518 multiplex quantification - APOA4 only samples_2018-05-18_12-13-20.sky.zip (265 MB)2019-05-311364864
20180518 multiplex quantification only samples_2018-05-18_11-58-07.sky.zip (380 MB)2019-05-3128357050268
20180509_prest-kinetics_2018-05-09_18-52-06.sky.zip (120 MB)2019-05-311613628684820
500 µE (PG-500uE_2ul_2019-05-16_15-23-09).sky.zip (7 MB)2019-05-292710510555811
Huidan-LHCSR3-Trypsin-PRM_FINAL2_2019-05-16_17-33-35.sky.zip (3 MB)2019-05-2920611225758
Huidan-LHCSR3-LysC-selected-tSIM-PRM_FINAL_2019-05-16_17-31-57.sky.zip (13 MB)2019-05-2931681366528
200µE (R5-8_V2_2019-05-16_15-31-29).sky.zip (8 MB)2019-05-2928979751616
matchedmatrixcalcurve_yeasthela_2019-05-28_13-23-23.sky.zip (1 GB)2019-05-283,22827,28927,28985,17542
matchedmatrixcalcurve_yeastwater_2019-05-24_12-06-14.sky.zip (1 GB)2019-05-243,73136,39336,39395,63942
20150514_Plasma_remove_points_2019-05-22_18-51-25.sky.zip (162 MB)2019-05-2220989862412
matchedmatrixcalcurve_csf_2019-05-22_14-34-12.sky.zip (531 MB)2019-05-224,00716,22916,22947,85630
matchedmatrixcalcurve_yeastyeast_2019-05-22_12-49-50.sky.zip (1 GB)2019-05-223,48230,60230,60290,48842
Janschitz et al_PRM_results_Panorama upload_2019-05-06_18-03-18.sky.zip (611 MB)2019-05-094475833712
LINCS_GCP_Plate76_annotated_minimized_2019-04-29_15-46-06.sky.zip (16 MB)2019-04-29818116287833
LINCS_P100_DIA_Plate51_annotated_minimized_2017-08-02_16-21-22.sky.zip (43 MB)2019-04-26909619292496
LINCS_P100_DIA_Plate41_annotated_minimized_2017-02-16_14-31-38.sky.zip (128 MB)2019-04-2690961921,00296
LINCS_P100_DIA_PlateAD2_annotated_minimized_2019-04-18_11-17-21.sky.zip (52 MB)2019-04-18909619278892
LINCS_P100_QC_PlateAD2_annotated_minimized_2019-04-18_11-15-52.sky.zip (6 MB)2019-04-18882413292
LINCS_GCP_Plate77_annotated_minimized_2019-04-17_09-22-00.sky.zip (19 MB)2019-04-17818116291233
LINCS_GCP_Plate75_annotated_minimized_2019-04-16_13-40-09.sky.zip (26 MB)2019-04-16818116291233
LINCS_GCP_Plate74_annotated_minimized_2019-04-16_13-21-11.sky.zip (24 MB)2019-04-16808016080533
LINCS_GCP_Plate73_annotated_minimized_2019-04-16_11-45-08.sky.zip (24 MB)2019-04-16818116289433
LINCS_P100_QC_Plate75_annotated_minimized_2019-04-08_16-33-18.sky.zip (4 MB)2019-04-08882415666
LINCS_P100_DIA_Plate75_annotated_minimized_2019-04-08_16-26-05.sky.zip (72 MB)2019-04-0890961922,76466
LINCS_P100_QC_Plate76_annotated_minimized_2019-04-08_15-34-30.sky.zip (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 peptides_2019-04-02_12-44-12.sky.zip (1 MB)2019-04-062221318
LINCS_P100_DIA_Plate73_annotated_minimized_2019-04-02_12-31-00.sky.zip (75 MB)2019-04-0290961922,76466
LINCS_P100_QC_Plate74a_annotated_minimized_2019-04-01_16-25-26.sky.zip (2 MB)2019-04-01882417436
LINCS_P100_DIA_Plate74a_annotated_minimized_2019-04-01_16-19-30.sky.zip (39 MB)2019-04-0190961922,76236
20181129_HFF1mut_IP_PRM_2019-03-15_14-54-27.sky.zip (4 MB)2019-04-0172020935
LINCS_P100_QC_Plate73_annotated_minimized_2019-03-28_10-49-18.sky.zip (5 MB)2019-03-28882416266
LINCS_P100_QC_Plate74_annotated_minimized_2019-03-27_16-36-40.sky.zip (6 MB)2019-03-27882417182
LINCS_P100_DIA_Plate74_annotated_minimized_2019-03-27_16-18-55.sky.zip (98 MB)2019-03-2790961922,76284
PRM_final_quantification_non-normalized_2019-03-18_10-30-44.sky.zip (148 MB)2019-03-265353514748
LINCS_P100_QC_Plate70_annotated_minimized_2019-03-13_14-12-46.sky.zip (7 MB)2019-03-13882416596
HLF with ladder_GUnames_2018-08-15_19-59-17.sky.zip (176 KB)2019-03-086045451
U87MG with ladder_GUnames_2018-08-15_20-02-38.sky.zip (429 KB)2019-03-08901181181
IgA with ladder_GUnames_2018-08-15_20-00-17.sky.zip (358 KB)2019-03-08801251251
HNE with ladder_GUnames_2018-08-15_19-59-46.sky.zip (174 KB)2019-03-086038381
PGM OG with ladder_GUnames_2018-08-15_20-01-34.sky.zip (370 KB)2019-03-08201061061
IgG with ladder_GUnames_2018-08-15_20-01-05.sky.zip (228 KB)2019-03-087061611
Fet NG with ladder_GUnames_2018-08-15_19-58-42.sky.zip (229 KB)2019-03-087063631
CBHI OG with ladder_GUnames_2018-08-15_19-58-20.sky.zip (93 KB)2019-03-082020201
CBHI NG with ladder_GUnames_2018-08-15_19-57-24.sky.zip (109 KB)2019-03-083028281
BLF with ladder_GUnames_2018-08-15_19-55-59.sky.zip (302 KB)2019-03-088085851
Fig3_U87MG_iRT_ladder_2018-08-15_19-50-07.sky.zip (429 KB)2019-03-08901191191
Fig5_SupFig3_Dextran_Spectral_Library_2018-08-15_19-53-02.sky.zip (40 KB)2019-03-08103181
Fig1_SupFig1_Ladder_tech_reps_2018-08-15_19-48-46.sky.zip (797 KB)2019-03-0810151510
Fig6_2019-01-26_23-48-44.sky.zip (63 MB)2019-03-088011634212
Fig2_SupFig2_iRT_linear.sky.zip (6 MB)2019-03-0820183216
PRM-Identifying the N-terminal amino acid for a TAG start codon_2019-03-04.sky.zip (3 MB)2019-03-05266583
PRM-Identifying the N-terminal amino acid for an ATG start codon_2019-03-04.sky.zip (1 MB)2019-03-05266563
Cortex_50_targets_PRM_finaL_20181212_2019-01-15_15-55-34.sky.zip (5 MB)2019-03-01551332582,2633
Pancreas_50_targets_PRM_final_20181210_2019-01-15_15-52-49.sky.zip (5 MB)2019-03-01631342602,0613
sasp_panel_ras_2019-02-26_13-24-23.sky.zip (51 MB)2019-02-2674730,02544,411576,8660
sasp_panel_irradiation_2019-02-26_13-00-53.sky.zip (17 MB)2019-02-261729,62414,598189,6470
MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments

  • Organism: Mouse
  • Instrument: TripleTOF 5600
  • SpikeIn: No
Abstract
Quantitative analysis of discovery-based proteomic work- flows now relies on high-throughput large-scale methods for identification and quantitation of proteins and post-translational modifications. Advancements in label-free quantitative techniques, using either data-dependent or data-independent mass spectrometric acquisitions, have coincided with improved instrumentation featuring greater precision, increased mass accuracy, and faster scan speeds. We recently reported on a new quantitative method called MS1 Filtering (Schilling et al. (2012) Mol. Cell. Proteomics 11, 202–214) for processing data-independent MS1 ion intensity chromatograms from peptide analytes using the Skyline software platform. In contrast, data- independent acquisitions from MS2 scans, or SWATH, can quantify all fragment ion intensities when reference spectra are available. As each SWATH acquisition cycle typically contains an MS1 scan, these two independent label-free quantitative approaches can be acquired in a single experiment. Here, we have expanded the capability of Skyline to extract both MS1 and MS2 ion intensity chromatograms from a single SWATH data-independent acquisition in an Integrated Dual Scan Analysis approach. The performance of both MS1 and MS2 data was examined in simple and complex samples using standard concentration curves. Cases of interferences in MS1 and MS2 ion intensity data were assessed, as were the differentiation and quantitation of phosphopeptide isomers in MS2 scan data. In addition, we demonstrated an approach for optimization of SWATH m/z window sizes to reduce interferences using MS1 scans as a guide. Finally, a correlation analysis was performed on both MS1 and MS2 ion intensity data obtained from SWATH acquisitions on a complex mixture using a linear model that automatically removes signals containing interferences. This work demonstrates the practical advantages of properly acquiring and processing MS1 precursor data in addition to MS2 fragment ion intensity data in a data-independent acquisition (SWATH), and provides an approach to simultaneously obtain independent measurements of relative peptide abundance from a single experiment. Molecular & Cellular Proteomics 14: 10.1074/mcp.O115.048181, 2015.
Experiment Description
Response Curves for a set of Acetylated Peptides in a Complex Matrix—Six lysine-acetylated synthetic peptides containing 13C615N2-Lys and 13C615N4-Arg were used to generate standard concentration curves in either a simple (25 fmol “six protein mix”) or a complex matrix (complex mitochondrial lysate, 0.3 μg on column), spanning from 4 attomoles to 25 femtomoles over 6 concentration points (0.004, 0.012, 0.037, 0.111, 0.333, 1, 3, and 25 fmol) for the following peptides: LVSSVSDLPKacR (HMGCS2 protein), MVQKacSLAR (HMGCS2 protein), AFVDSCLQLHETKacR (LCAD protein), YAPVAKacDLASR (SDHA protein), LFVDKacIR (ATP5J protein), and AFGGQSLKacFGK (SDHA protein). Three replicate concentration curves, each with injections from lowest to highest spike concentration were acquired on the TripleTOF 5600 (SWATH MS2 mode). Mouse Liver Mitochondrial Protein Lysate—Mitochondria were isolated by differential centrifugation from liver WT (C57BL/6) mice, and proteins were denatured with 1% dodecyl-maltoside and 10 M urea. Samples were then diluted 1:10, reduced with 4.5 mM TCEP (37°C for 1 h), alkylated with 10 mM iodoacetamide (30 min at RT in the dark), and incubated overnight at 37°C with sequencing grade trypsin added at a 1:50 enzyme:substrate ratio (wt/wt). Samples were then acidified with formic acid and desalted using HLB Oasis SPE cartridges. Samples were eluted, concentrated to near dryness, and resuspended prior to analysis. Samples were processed in duplicates and three injection replicates at a concentration of 100 ng or 33 ng were acquired in a randomized order on the TripleTOF 5600 mass spectrometer. PHDE1α Kinase Inhibitor Study—Mouse liver mitochondria from wild-type mice (C57BL/6) were isolated as described previously (29, 30). Mitochondria (1 mg) were incubated at room temperature with 5 mM DCA (10 mM Hepes pH 7.2) for 0, 5, 10, 30, 60, and 120 min. Samples were digested with trypsin and phosphopeptides were enriched by IMAC chromatography as previously described (11). Heavy phosphopeptides for pSer-293 (YHGHS293MSDPGVSYR[13C615N4]) and pSer-300 (YHGHSMS300DPGVSYR[13C615N4]) were spiked at 25 fmol into each sample. Equal volumes of eluted phosphopeptides were desalted using C-18 zip-tips and then analyzed on the TripleTOF 5600.
Sample Description
Data sets acquired on a TripleTOF 5600 mass spectrometer: 1) MS1 and SWATH-MS – Kinase Inhibitor Study for quantifying changes in endogenous phosphopeptide isomers YHGHpS293MSDPGVSYR and YHGHSMSDPGVpS300YR from PDHE1a (3 injection replicates), go to Kinase Inhibitor Study in Panorama, download Kinase Inhibitor Study. 2) MS1 and SWATH-MS – dilution curve of heavy labeled phosphopeptide isomers YHGHpS293MSDPGVSYR and YHGHSMSDPGVpS300YR from PDHE1a (3 injection replicates), download various isomer ratio sub files: 16:1/1:16, 8:1/1:8, 4:1/1:4, 1:2, 1:1. 3) MS1 and SWATH-MS – Optimization of SWATH window size (25 and 10 m/z, respectively) to reduce interferences in a mitochondrial lysate (5 injection replicates), go to SWATH Optimization Study in Panorama, download SWATH Optimization Study. 4) MS1 and SWATH-MS – MS1 and MS2 Data Processing Study for removal of interferences using mitochondrial lysates at a concentration of 100 ng or 33 ng (2 process replicates, 3 injection replicates), go to Data Processing Study in Panorama, download Data Processing Study (sample legend).
Created on 6/18/15, 9:58 PM