Dataset Information

Human breast cancer cell line LC-MS/MS

ABSTRACT: Human breast cancer cell lines were pooled according to ER/Her2 status. The whole cell lysate samples were reduced and treated with iodoacetamide and then digested with trypsin. The samples were separated into 6 fractions by online high pH reverse-phase fractionation and then analyzed by reverse-phase chromatography coupled to a Thermo LTQ-Orbitrap Velos mass spectrometer. MS/MS files were searched against version 3.69 of the Human International Protein Index (IPI) sequence database with decoy sequences by the X!Tandem database search engine with k-score plugin with the following parameters: tryptic enzyme constraint allowing for up to two missed cleavages. Peptide MH+ mass tolerances set at 2.0 Da with post search filtering of precursor mass to 50 ppm. Oxidized methionine as a variable modification and carbamidomethylated cysteine as a static modification. FDR < 0.005 based on decoy search.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jacob Kennedy

PROVIDER: PXD000246 | Pride | 2013-10-18

REPOSITORIES: Pride

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Publications

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

Kennedy Jacob J JJ Abbatiello Susan E SE Kim Kyunggon K Yan Ping P Whiteaker Jeffrey R JR Lin Chenwei C Kim Jun Seok JS Zhang Yuzheng Y Wang Xianlong X Ivey Richard G RG Zhao Lei L Min Hophil H Lee Youngju Y Yu Myeong-Hee MH Yang Eun Gyeong EG Lee Cheolju C Wang Pei P Rodriguez Henry H Kim Youngsoo Y Carr Steven A SA Paulovich Amanda G AG

Nature methods 20131208 2

Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays rep ...[more]

PMID: 24317253

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Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.

Project description:Tistlia consotensis is a halotolerant Rhodospirillaceae that was isolated from a saline spring located in the Colombian Andes with a salt concentration close to seawater (4.5%w/vol). We cultivated this microorganism in three NaCl concentrations, i.e. optimal (0.5%), without (0.0%) and high (4.0%) salt concentration, and analyzed its cellular proteome. For assigning tandem mass spectrometry data, we first sequenced its genome and constructed a six reading frame ORF database from the draft sequence. We annotated only the genes whose products (872) were detected. We compared the quantitative proteome data sets recorded for the three different growth conditions.Peak lists were generated with the MASCOT DAEMON software (version 2.3.2) from Matrix Science using the extract_msn.exe data import filter from the Xcalibur FT package (version 2.0.7) proposed by ThermoFisher. Data import filter options were set at 400 (minimum mass), 5,000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1,000 (threshold). MS/MS spectra were searched against the home-made ORF database with the following parameters: tryptic peptides with a maximum of 2 miss cleavages during proteolytic digestion, a mass tolerance of 5 ppm on the parent ion and 0.5 Da on the MS/MS, fixed modification for carbamidomethylated Cys (+57.0215) and variable modification for oxidized Met (+15.9949). All peptide matches with a peptide score above its query threshold set at p < 0.05 with the ORF database and rank 1 were parsed using the IRMa 1.28.0 software. False-positive rate for peptide identification was estimated using a decoy database as below 0.5% with these parameters. MS/MS spectra assigned to several loci were systematically removed. A protein was considered validated when at least two different peptides were detected in the same experiment. False-positive identification of proteins was estimated using a reverse decoy database as below 0.1% with these parameters The number of MS/MS spectra per protein (spectral counts) was determined for the three replicates in each growth condition. The protein abundances were compared using the T-Fold option of the PatternLab 2.0 software. This module allows normalising the spectral count datasets, calculating the average fold changes with statistics (t-test), and estimating the resulting theoretical false discovery rate. Stringent parameters were used in this analysis: minimum fold change of 1.5, minimum p-value of 0.05 and BH-FDR Alfa of 0.15.

Dataset Information

Human breast cancer cell line LC-MS/MS

Publications

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

Similar Datasets

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