Inference and quantification of peptidoforms in large sample cohorts by SWATH-MS: Phosphopeptide-enriched data set
This data set represent an experiment comparing enriched phosphopeptides of a human U2OS cell line. Phosphopeptide-enriched samples of ten nocodazole treated and ten control U2OS cell line samples were acquired each in DDA and DIA (SWATH-MS) modes.
Sample Processing Protocol
DDA: The samples were measured on an AB Sciex 6600 TripleTOF mass spectrometer operated in DDA mode. The mass spectrometer was interfaced with an Eksigent NanoLC Ultra 2D Plus HPLC system. Peptides were directly injected onto a 20-cm PicoFrit emitter (New Objective, self-packed to 20 cm with Magic C18 AQ 3-μm 200-Å material), and then separated using a 120- min gradient from 2–30% (buffer A 0.1% (v/v) formic acid, 2% (v/v) acetonitrile, buffer B 0.1% (v/v) formic acid, 90% (v/v) acetonitrile) at a flow rate of 300 nL/min. MS1 spectra were collected in the range 360–1,460 m/z for 500 ms. The 20 most intense precursors with charge state 2–5 which exceeded 250 counts per second were selected for fragmentation, and MS2 spectra were collected in the range 50–2,000 m/z for 150 ms. The precursor ions were dynamically excluded from reselection for 20 s. DIA: The samples were measured in SWATH-MS mode on the same LC-MS/MS systems used for DDA measurements. In SWATH-MS mode the SCIEX 6600 TripleTOF instrument was specifically tuned to optimize the quadrupole settings for the selection of 64 variable wide precursor ion selection windows. The 64-variable window schema was optimized based on a normal human cell lysate sample, covering the precursor mass range of 400–1,200 m/z. The effective isolation windows can be considered as being 399.5~408.2, 407.2~415.8, 414.8~422.7, 421.7~429.7, 428.7~437.3, 436.3~444.8, 443.8~451.7, 450.7~458.7, 457.7~466.7, 465.7~473.4, 472.4~478.3, 477.3~485.4, 484.4~491.2, 490.2~497.7, 496.7~504.3, 503.3~511.2, 510.2~518.2, 517.2~525.3, 524.3~533.3, 532.3~540.3, 539.3~546.8, 545.8~554.5, 553.5~561.8, 560.8~568.3, 567.3~575.7, 574.7~582.3, 581.3~588.8, 587.8~595.8, 594.8~601.8, 600.8~608.9, 607.9~616.9, 615.9~624.8, 623.8~632.2, 631.2~640.8, 639.8~647.9, 646.9~654.8, 653.8~661.5, 660.5~670.3, 669.3~678.8, 677.8~687.8, 686.8~696.9, 695.9~706.9, 705.9~715.9, 714.9~726.2, 725.2~737.4, 736.4~746.6, 745.6~757.5, 756.5~767.9, 766.9~779.5, 778.5~792.9, 791.9~807, 806~820, 819~834.2, 833.2~849.4, 848.4~866, 865~884.4, 883.4~899.9, 898.9~919, 918~942.1, 941.1~971.6, 970.6~1006, 1005~1053, 1052~1110.6, 1109.6~1200.5 (including 1 m/z window overlapping). SWATH MS2 spectra were collected from 300 to 2,000 m/z. The collision energy (CE) was optimized for each window according to the calculation for a charge 2+ ion centered upon the window with a spread of 15 eV. An accumulation time (dwell time) of 50 ms was used for all fragment-ion scans in high-sensitivity mode and for each SWATH-MS cycle a survey scan in high- resolution mode was also acquired for 200 ms. Per MS injection 2 μg of protein amount was loaded onto the HPLC column.
Data Processing Protocol
Assay library generation using DDA data: All raw data was analyzed in a combined setting with MaxQuant (22.214.171.124) using primarily the default parameters: The non-redundant reviewed human protein FASTA was obtained from the UniProtKB/Swiss-Prot72 (2016-12-19) and appended with iRT peptide sequences and searched with static C (Carbamidomethyl), variable M (Oxidation) and variable STY (Phospho) modifications. “Match-between-runs” and the MaxLFQ algorithm were enabled. All specific parameters are provided in the file mqpar.xml in the ProteomeXchange repository. To derive peptide query parameters, we selected the best scoring spectrum per peptidoform as reported by Andromeda in the file “msms.txt”. RT calibration was conducted using the spiked-in iRT-kit per run. OpenSwathAssayGenerator and OpenSwathDecoyGenerator (OpenMS 2.1) were run as described above. For all other analyses, we used the reported confidence values and intensities from the file “Phospho (STY)Sites.txt”. OpenSWATH / PyProphet: OpenSwathWorkflow (OpenMS 2.1) was run with the following parameters -min_upper_edge_dist 1 - mz_extraction_window 0.05 -rt_extraction_window 600 - extra_rt_extraction_window 100 -min_rsq 0.95 -min_coverage 0.6 - use_ms1_traces -enable_uis_scoring -Scoring:uis_threshold_peak_area 0 - Scoring:uis_threshold_sn -1 -Scoring: stop_report_after_feature 5 -tr_irt hroest_DIA_iRT.TraML. The following subset of scores was used on MS2-level: xx_swath_prelim_score library_corr yseries_score xcorr_coelution_weighted massdev_score norm_rt_score library_rmsd bseries_score intensity_score xcorr_coelution log_sn_score isotope_overlap_score massdev_score_weighted xcorr_shape_weighted isotope_correlation_score xcorr_shape. All MS1 and UIS scores were used for pyprophet. pyprophet was run individually on all files with the following parameters: --final_statistics.emp_p --qvality.enable --qvality.generalized -- ms1_scoring.enable --uis_scoring.enable --semi_supervised_learner.num_iter=20 --xeval.num_iter=20 --ignore.invalid_score_columns. TRIC was run with the following parameters: feature_alignment.py: --file_format openswath --fdr_cutoff 0.01 --max_fdr_quality 0.2 --mst:useRTCorrection True --mst:Stdev_multiplier 3.0 --method LocalMST --max_rt_diff 30 --alignment_score 0.0001 --frac_selected 0 --realign_method lowess_cython --disable_isotopic_grouping
George Rosenberger, Columbia University
Ruedi Aebersold, ETH Zurich Prof. Dr. Ruedi Aebersold Institute of Molecular Systems Biology Head of Department of Biology HPT E 78 Auguste-Piccard-Hof 1 CH-8093 Zurich Switzerland ( lab head )
Rosenberger G, Liu Y, Röst HL, Ludwig C, Buil A, Bensimon A, Soste M, Spector TD, Dermitzakis ET, Collins BC, Malmström L, Aebersold R. Inference and quantification of peptidoforms in large sample cohorts by SWATH-MS. Nat Biotechnol. 2017 Jun 12 PubMed: 28604659