| Metadata |
| datasetIdentifier | PASS00391 |
| datasetType | MSMS |
| submitter | Maria Romina Girotti <romina.girotti@cruk.manchester.ac.uk> |
| submitter_organization | Cancer research UK Manchester Institute |
| lab_head_full_name | Prof. Richard Marais |
| lab_head_email | richard.maraiscruk.manchester.ac.uk |
| lab_head_organization | Cancer research UK Manchester Institute |
| lab_head_country | United Kingdom |
| datasetTag | SanchezLaorden2014 |
| datasetTitle | Secretome analysis - ITRAQ quant |
| publicReleaseDate | 2014-01-15 00:00:00 |
| finalizedDate | 2014-01-15 12:12:43 |
| summary | WM1791c cells were seeded at 80% confluence in 150 mm-plates, grown for 24 hours, washed three times with PBS, and incubated in serum-free medium for additional 24 hours in the presence of vehicle (DMSO) or PLX4720 (1 µM). Conditioned media were collected and cleared from cell and debris by centrifugation. Resulting supernatants were supplemented with 0.01 µM aprotinine, 1 µM leupeptin) (Sigma) and concentrated 60-fold in Centriprep-3 (Millipore). Proteins were then precipitated with 10% TCA/acetone and the resulting pellet was solubilized in standard cell Lysis Buffer (8M urea). Protein concentration was determined using the BCA assay (Sigma). |
| contributors | Berta Sanchez-Laorden, Amaya Viros, Maria Romina Girotti, Malin Pedersen, Grazia Saturno, Alfonso Zambon, Dan Niculescu-Duvaz, Samra Turajlic, Andrew Hayes, Martin Gore, James Larkin, Paul Lorigan, Martin Cook, Caroline Springer and Richard Marais. |
| publication | Berta Sanchez-Laorden, Amaya Viros, Maria Romina Girotti, Malin Pedersen, Grazia Saturno, Alfonso Zambon, Dan Niculescu-Duvaz, Samra Turajlic, Andrew Hayes, Martin Gore, James Larkin, Paul Lorigan, Martin Cook, Caroline Springer and Richard Marais.
Title: BRAF inhibitors induce metastasis in RAS-mutant and inhibitor-resistant melanoma cells through MEK/ERK pathway reactivation
Manuscript submitted to Science Signaling. |
| growth | Human cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS 1% penicillin/streptomycin. Mouse and human tumor derived cell lines obtained were cultured in DMEM/10% FBS/1% penicillin/streptomycin and 0.1mg/ml primocin (InvivoGen). |
| treatment | WM1791c cells were seeded at 80% confluence in 150 mm-plates, grown for 24 hours, washed three times with PBS, and incubated in serum-free medium for additional 24 hours in the presence of vehicle (DMSO) or PLX4720 (1 µM). Conditioned media were collected. |
| extraction | Conditioned media were collected and cleared from cell and debris by centrifugation. Resulting supernatants were supplemented with 0.01 µM aprotinine, 1 µM leupeptin) (Sigma) and concentrated 60-fold in Centriprep-3 (Millipore). Proteins were then precipitated with 10% TCA/acetone and the resulting pellet was solubilized in standard cell Lysis Buffer (8M urea). Protein concentration was determined using the BCA assay (Sigma). |
| separation | Protein samples diluted in 8 M urea were subsequently reduced t2 M using 100 mM ammonium acetate pH 8 before tryptic digestion. Proteins were reduced with 10 mM dithiothreitol for 30 min at 56ºC, then alkylated with 55 mM iodoacetamide for 1 hour at room temperature in the dark. Cell lysates were diluted to a final urea concentration of 1.6 M with 50 mM ammonium bicarbonate, and digested with trypsin (substrate:enzyme = 50) at 37ºC overnight with end-over-end rotation. The resulting peptide solutions were acidified with 10% TFA (trifluoroacetic) and desalted on a Waters C18 solid phase extraction plate. Eluted peptides were divided into 100 µg aliquots, lyophilized to complete dryness, and stored at -80ºC until needed. |
| digestion | iTRAQ labelling. For DMSO and PLX4720 treated samples, duplicates were performed such that peptides were independently labeled and analyzed by LC- MS/MS twice. Desalted peptides were labeled with iTRAQ reagents (43) according to the manufacturer’s instructions. Briefly, 100 µg aliquots of dried peptides were reconstituted in 30 µL 0.5 M triethylammonium bicarbonate. One tube of iTRAQ reagent (ABSciex) was reconstituted in 70 µl ethanol and added to each peptide solution. The reaction was allowed to proceed for 1 h at room temperature. Derivatized peptides were combined dried by vacuum centrifugation, and desalted on a Waters C18 solid phase extraction plate. iTRAQ labeled peptides were lyophilized to complete dryness and stored at -80ºC until needed. Secretome samples were labeled with iTRAQ 4plex reagent (114-Vehicle 1, 115-PLX4720 1, 116-1PLX4720
1, or 117- Vehicle). |
| acquisition | LC-MS/MS. Reversed phase chromatography was performed using an HP1200 platform (Agilent). Forty percent of samples were analysed as 4 µl injection. Peptides were resolved on a 75 mm I.D. C18 Pepmap column (3 µm particle size; LC Packings/Dionex) a range of linear gradients of 96:4 to 50:50 buffer A:B (buffer A:2% acetonitrile/0.1% formic acid; buffer B: 80% acetonitrile/0.1% formic acid) at 300 nL/min were used for specific sample analyses. Peptides were ionised by electrospray ionisation using 1.5 kV applied directly to the post-column LC eluent via a microtee built into the nanospray source. Sample was infused into an LTQ Velos Orbitrap mass spectrometer (Thermo Fisher Scientific) using a non-coated SilicaTip emitter (20 µm I.D., 10 µm tapered tip; New Objectives). The ion transfer tube was heated to 200°C and the S-lens set to 60%. MS/MS were acquired using data dependent acquisition to sequence the top 10 most intense ions using enhanced ion trap scans. Automatic gain control was set to 1,000,000 for FT-MS and 30,000 for IT-MS/MS, full FT-MS maximum inject time was 500 ms and normalized collision energy was set to 35% with an activation time of 10 ms. Wideband activation was used to co-fragment precursor ions undergoing neutral loss of up to -20 m/z from the parent ion, including loss of water/ammonia. MS/MS was acquired with a 10 ppm mass window for either15s based on a maximum exclusion list of 500 entries.
Data processing. The iTRAQ-labeled peptides were fragmented under CID conditions to display reporter ions at m/z 114.1 and 117.1. The ratios of the peak area of the iTRAQ reporter ions represent the relative abundance of the peptides in the samples, which were automatically quantified with Proteome Discoverer v1.2. For
this purpose, raw MS/MS data was submitted for database searching using Proteome Discoverer v1.2 and Mascot v2.2. MS/MS spectra from each acquisition were extracted and converted into .mgf files, and then searched against SwissProt database.57.14 (514,789 sequences) using Mascot. Search parameters included trypsin specificity with up to 2 missed cleavages, fixed carbamidomethylation on cysteine, fixed iTRAQ modification on N-terminus and lysine, variable deamidation on asparagine and glutamine, variable oxidation on methionine and variable phosphorylation on serine, threonine and tyrosine. MS/MS-based peptide and protein identifications were grouped and validated using Scaffold v3.0 (Proteome Software Inc.). Protein identifications were automatically accepted if they contained at least 2 unique peptides assigned with at least 95% confidence by Peptide Prophet.
Data analysis. For iTRAQ quantification, the ratios of iTRAQ reporter ion intensities in MS/MS spectra (m/z 114.11-117.11) from raw data sets were used to calculate fold changes between samples. Ratios were derived by Proteome Discoverer version 1.2. False discovery rate (FDR) for peptide search is a statistical value that determines the number of false identifications among all identifications. Proteome Discoverer calculates the percentage of false identifications using a separate decoy database (reverse database) that contains the reversed sequences of the protein entries. An FDR threshold of 1% was used in this study. Precursor and reporter ion window tolerance were fixed at 5 ppm and 0.05 Da, respectively. The criteria specified for generation of peak lists include signal to noise ratio of 1.5 and inclusion of precursor mass range of 600–8000 Da. Proteome Discoverer software performs automated statistical analysis of the results and uses unique peptides to calculate accurate relative protein quantification. The relative protein abundance ratios were then determined with respect to the control sample (vehicle) (tag 114 or 117). The ratios of all peptides corresponding to the same proteins were averaged. The resulting protein ratios were again normalized by their population median. Two technical replicates were included in each experiment (114=control; 115=PLX; 116=control and 117=PLX) and two biological replicates were analysed. The ratios 115/114 and 117/116 were averaged. A fold change cut-off of 1.5 was set to identify molecules whose expression was differentially regulated. |
| informatics | MS files interrogated using Mascot via Proteome Discoverer 1.4 as follows:
Search report summary of file(s):
- E:\Data\PR150\10993.msf
Created with Discoverer version: 1.4.0.288
================================================================================
Number of filtered/unfiltered result items:
- 472/483 protein group(s)
- 1001/1001 merged protein(s)
- 3674/3674 peptide(s)
- 9275/9275 PSM(s)
- 22420/22420 search input(s)
================================================================================
Peptide Grouping Options
- Show peptide groups: True
- Group peptides by: Mass and Sequence
Protein Grouping Options
- Enable protein grouping: True
- Consider leucine and isoleucine as equal: True
- Consider only PSMs with confidence at least: Medium
- Consider only PSMs with delta Cn better than: 0.15
- Apply strict maximum parsimony principle: True
No filters applied for data reduction
No result filters applied
================================================================================
Summary of file E:\Data\PR150\10993.msf
Workflow created with Discoverer version: 1.4.0.288 (DBVersion:79)
================================================================================
Search name: 10993
Search description: -
Search date: 01/14/2014 11:20:57
================================================================================
The pipeline tree:
------------------
|-(0) Spectrum Files
|-(1) Spectrum Selector
|-(2) Scan Event Filter
|-(11) Mascot
|-(8) Percolator
|-(6) Reporter Ions Quantifier
------------------------------------------------------------------------------
Processing node 0: Spectrum Files
------------------------------------------------------------------------------
Input Data:
-----------------------------
File Name(s): E:\Data\PR150\10993.raw
------------------------------------------------------------------------------
Processing node 1: Spectrum Selector
------------------------------------------------------------------------------
1. General Settings:
-----------------------------
Precursor Selection: Use MS1 Precursor
Use New Precursor Reevaluation: True
2. Spectrum Properties Filter:
-----------------------------
Lower RT Limit: 0
Upper RT Limit: 0
First Scan: 0
Last Scan: 0
Lowest Charge State: 2
Highest Charge State: 5
Min. Precursor Mass: 700 Da
Max. Precursor Mass: 6000 Da
Total Intensity Threshold: 0
Minimum Peak Count: 1
3. Scan Event Filters:
-----------------------------
MS Order: Is MS2
Min. Collision Energy: 0
Max. Collision Energy: 100
Scan Type: Is Full
4. Peak Filters:
-----------------------------
S/N Threshold (FT-only): 3
5. Replacements for Unrecognized Properties:
-----------------------------
Unrecognized Charge Replacements: Automatic
Unrecognized Mass Analyzer Replacements: ITMS
Unrecognized MS Order Replacements: MS2
Unrecognized Activation Type Replacements: CID
Unrecognized Polarity Replacements: +
6. Just for Testing:
-----------------------------
Precursor Clipping Range Before: 2.5 Da
Precursor Clipping Range After: 5.5 Da
------------------------------------------------------------------------------
Processing node 2: Scan Event Filter
------------------------------------------------------------------------------
Filter Settings:
-----------------------------
Mass Analyzer: Any
MS Order: Is MS2
Activation Type: Any
Min. Collision Energy: 0
Max. Collision Energy: 100
------------------------------------------------------------------------------
Processing node 11: Mascot
------------------------------------------------------------------------------
1. Input Data:
-----------------------------
Protein Database: SwissProt
Enzyme Name: Trypsin
Maximum Missed Cleavage Sites: 2
Instrument: ESI-TRAP
Taxonomy: . . . . . . . . . . . . . . . . Homo sapiens (human)
1.1 Peptide Scoring Options:
-----------------------------
Peptide Cut Off Score: 10
Peptide Without Protein Cut Off Score: 5
1.2 Protein Scoring Options:
-----------------------------
Use MudPIT Scoring: Automatic
Protein Relevance Threshold: 20
Protein Relevance Factor: 1
2. Tolerances:
-----------------------------
Precursor Mass Tolerance: 5 ppm
Fragment Mass Tolerance: 0.25 Da
Use Average Precursor Mass: False
3. Modification Groups:
-----------------------------
From Quan Method: None
4. Dynamic Modifications:
-----------------------------
1. Dynamic Modification: Acetyl (Protein N-term)
2. Dynamic Modification: iTRAQ4plex (N-term)
3. Dynamic Modification: Gln->pyro-Glu (N-term Q)
4. Dynamic Modification: Oxidation (M)
5. Dynamic Modification: iTRAQ4plex (K)
6. Dynamic Modification:
5. Static Modifications:
-----------------------------
1. Static Modification: Carbamidomethyl (C)
------------------------------------------------------------------------------
Processing node 8: Percolator
------------------------------------------------------------------------------
1. Input Data:
-----------------------------
Maximum Delta Cn: 0.05
2. Decoy Database Search:
-----------------------------
Target FDR (Strict): 0.01
Target FDR (Relaxed): 0.05
Validation based on: q-Value
------------------------------------------------------------------------------
Processing node 6: Reporter Ions Quantifier
------------------------------------------------------------------------------
1. Quantification Method:
-----------------------------
Quantification Method: iTRAQ 4plex (Custom)
2. Peak Integration:
-----------------------------
Integration Tolerance: 20 ppm
Integration Method: Most Confident Centroid
3. Scan Event Filters:
-----------------------------
Mass Analyzer: FTMS
MS Order: MS2
Activation Type: HCD
Min. Collision Energy: 0
Max. Collision Energy: 100
================================================================================
Further information:
Mascot (11):
- Fasta database information: FASTA db: SwissProt_040511a.fasta
Version: 2.3
Number of sequences: 526969
Number of sequences after taxonomy: 20305
Taxonomy:
Enzyme:
Number of queries: 22420
- MudPitScoring: True
Percolator (8):
- Percolator Output: Results for Mascot (11):
Iteration 1 : After the iteration step, 4994 target PSMs with q<0.01 were estimated by cross validation
Iteration 2 : After the iteration step, 5247 target PSMs with q<0.01 were estimated by cross validation
Iteration 3 : After the iteration step, 5323 target PSMs with q<0.01 were estimated by cross validation
Iteration 4 : After the iteration step, 5342 target PSMs with q<0.01 were estimated by cross validation
Iteration 5 : After the iteration step, 5338 target PSMs with q<0.01 were estimated by cross validation
Iteration 6 : After the iteration step, 5341 target PSMs with q<0.01 were estimated by cross validation
Iteration 7 : After the iteration step, 5343 target PSMs with q<0.01 were estimated by cross validation
Iteration 8 : After the iteration step, 5350 target PSMs with q<0.01 were estimated by cross validation
Iteration 9 : After the iteration step, 5351 target PSMs with q<0.01 were estimated by cross validation
Iteration 10 : After the iteration step, 5353 target PSMs with q<0.01 were estimated by cross validation
Feature name Raw weight Normalized weight
IonScore 0.0693 1.3
Delta Cn From Second PSM 2.4634 0.6592
Binomial Score 0.0351 2.6008
Isolation Interference [%] -0.0119 -0.1827
MH+ [Da] 0.0013 0.8349
Delta Mass [Da] -10.8572 -0.0493
Delta Mass [ppm] -0.4457 -1.1097
Absolute Delta Mass [Da] 113.0508 0.3317
Absolute Delta Mass [ppm] -1.1477 -1.6421
Peptide Length -0.0644 -0.3631
Is z=1 0 0
Is z=2 -0.7847 -0.3915
Is z=3 0.387 0.191
Is z=4 0.8599 0.2577
Is z=5 1.7874 0.2063
Is z>5 0 0
# Missed Cleavages -1.2715 -0.9947
Log Peptides Matched 0 0
Log Total Intensity -0.6944 -0.5003
Fraction Matched Intensity [%] 0.002 0.0344
Fragment Coverage Series A, B, C [%] -0.0335 -0.8506
Fragment Coverage Series X, Y, Z [%] -0.0096 -0.2639
Log Matched Fragment Series Intensities A, B, C -0.017 -0.049
Log Matched Fragment Series Intensities X, Y, Z -0.0183 -0.0513
Longest Sequence Series A, B, C 0.2919 0.6857
Longest Sequence Series X, Y, Z 0.3665 1.0401
IQR Fragment Delta Mass [Da] -5.2391 -0.3353
IQR Fragment Delta Mass [ppm] -0.0003 -0.0401
Mean Fragment Delta Mass [Da] -7.8013 -0.3219
Mean Fragment Delta Mass [ppm] 0.0021 0.2191
Mean Absolute Fragment Delta Mass [Da] -2.5685 -0.1069
Mean Absolute Fragment Delta Mass [ppm] 0.0029 0.2916
m0 -3.356 -2.8145
================================================================================
Processing details:
01/14/2014 12:24 PM (6):Reporter Ions Quantifier: Saved quantification results for 11595 spectra.
01/14/2014 12:22 PM (11):Mascot: Total search time was 4 min 50 s.
01/14/2014 12:22 PM (8):Percolator: Performing percolator for Mascot (11) took 3 min 32 s.
01/14/2014 12:22 PM (11):Mascot: Search completed
01/14/2014 12:22 PM (11):Mascot: 1001 protein(s) + 397 decoy proteins scored and inserted into result file in 2 s.
01/14/2014 12:22 PM (11):Mascot: 1001 protein(s) scored
01/14/2014 12:22 PM (11):Mascot: Search result finalization started.
01/14/2014 12:22 PM (8):Percolator: Start reading Percolator results
01/14/2014 12:22 PM (8):Percolator: Processing took 21.08 cpu seconds or 21 seconds wall time
01/14/2014 12:22 PM (8):Percolator: PSMId score q-value posterior_error_prob peptide proteinIds
01/14/2014 12:22 PM (8):Percolator: Calibrating statistics - calculating Posterior error probabilities (PEPs)
01/14/2014 12:22 PM (8):Percolator: New pi_0 estimate on merged list gives 1623 peptides over q=0.0100
01/14/2014 12:22 PM (8):Percolator: Calibrating statistics - calculating q values
01/14/2014 12:22 PM (8):Percolator: Selecting pi_0=0.7075
01/14/2014 12:22 PM (8):Percolator: Tossing out "redundant" PSMs keeping only the best scoring PSM for each unique peptide.
01/14/2014 12:22 PM (8):Percolator: Merging results from 3 datasets
01/14/2014 12:22 PM (8):Percolator: Found 5224 target PSMs scoring over 1.0000% FDR level on testset
01/14/2014 12:22 PM (8):Percolator: 0.0693 2.4634 0.0351 -0.0119 0.0013 -10.8572 -0.4457 113.0508 -1.1477 -0.0644 0.0000 -0.7847 0.3870 0.8599 1.7874 0.0000 -1.2715 0.0000 -0.6944 0.0020 -0.0335 -0.0096 -0.0170 -0.0183 0.2919 0.3665 -5.2391 -0.0003 -7.8013 0.0021 -2.5685 0.0029 -3.3560
01/14/2014 12:22 PM (8):Percolator: 1.3 0.6592 2.6008 -0.1827 0.8349 -0.0493 -1.1097 0.3317 -1.6421 -0.3631 0.0000 -0.3915 0.1910 0.2577 0.2063 0.0000 -0.9947 0.0000 -0.5003 0.0344 -0.8506 -0.2639 -0.0490 -0.0513 0.6857 1.0401 -0.3353 -0.0401 -0.3219 0.2191 -0.1069 0.2916 -2.8145
01/14/2014 12:22 PM (8):Percolator: IonScore Delta Cn From Second PSM Binomial Score Isolation Interference [%] MH+ [Da] Delta Mass [Da] Delta Mass [ppm] Absolute Delta Mass [Da] Absolute Delta Mass [ppm] Peptide Length Is z=1 Is z=2 Is z=3 Is z=4 Is z=5 Is z>5 # Missed Cleavages Log Peptides Matched Log Total Intensity Fraction Matched Intensity [%] Fragment Coverage Series A, B, C [%] Fragment Coverage Series X, Y, Z [%] Log Matched Fragment Series Intensities A, B, C Log Matched Fragment Series Intensities X, Y, Z Longest Sequence Series A, B, C Longest Sequence Series X, Y, Z IQR Fragment Delta Mass [Da] IQR Fragment Delta Mass [ppm] Mean Fragment Delta Mass [Da] Mean Fragment Delta Mass [ppm] Mean Absolute Fragment Delta Mass [Da] Mean Absolute Fragment Delta Mass [ppm] m0
01/14/2014 12:22 PM (8):Percolator: # first line contains normalized weights, second line the raw weights
01/14/2014 12:22 PM (8):Percolator: Obtained weights (only showing weights of first cross validation set)
01/14/2014 12:22 PM (8):Percolator: Iteration 10 : After the iteration step, 5353 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 9 : After the iteration step, 5351 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 8 : After the iteration step, 5350 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 7 : After the iteration step, 5343 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 6 : After the iteration step, 5341 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 5 : After the iteration step, 5338 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 4 : After the iteration step, 5342 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 3 : After the iteration step, 5323 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 2 : After the iteration step, 5247 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: Iteration 1 : After the iteration step, 4994 target PSMs with q<0.01 were estimated by cross validation
01/14/2014 12:22 PM (8):Percolator: ---Training with Cpos selected by cross validation, Cneg selected by cross validation, fdr=0.01
01/14/2014 12:22 PM (8):Percolator: Reading in data and feature calculation took 24.305 cpu seconds or 25 seconds wall time
01/14/2014 12:22 PM (8):Percolator: Estimating 3847 over q=0.01 in initial direction
01/14/2014 12:22 PM (8):Percolator: Selected feature number 3 as initial search direction, could separate 2508 positives in that direction
01/14/2014 12:22 PM (8):Percolator: Selected feature number 3 as initial search direction, could separate 2608 positives in that direction
01/14/2014 12:22 PM (8):Percolator: Selected feature number 3 as initial search direction, could separate 2580 positives in that direction
01/14/2014 12:22 PM (8):Percolator: selecting cneg by cross validation
01/14/2014 12:22 PM (8):Percolator: selecting cpos by cross validation
01/14/2014 12:22 PM (8):Percolator: Train/test set contains 12113 positives and 11068 negatives, size ratio=1.09442 and pi0=1
01/14/2014 12:21 PM (8):Percolator: e6e22773-e9a6-4a26-9694-1ca77a797099 Delta Cn From Second PSM Binomial Score b8754504-e95e-476b-b9a4-454d4bb53aeb 1d91a87b-953a-4887-9f22-f75a497a3538 Delta Mass [Da] Delta Mass [ppm] Absolute Delta Mass [Da] Absolute Delta Mass [ppm] Peptide Length Is z=1 Is z=2 Is z=3 Is z=4 Is z=5 Is z>5 041eb6d5-e486-44a0-9bc1-19e25811c686 Log Peptides Matched Log Total Intensity Fraction Matched Intensity [%] Fragment Coverage Series A, B, C [%] Fragment Coverage Series X, Y, Z [%] Log Matched Fragment Series Intensities A, B, C Log Matched Fragment Series Intensities X, Y, Z Longest Sequence Series A, B, C Longest Sequence Series X, Y, Z IQR Fragment Delta Mass [Da] IQR Fragment Delta Mass [ppm] Mean Fragment Delta Mass [Da] Mean Fragment Delta Mass [ppm] Mean Absolute Fragment Delta Mass [Da] Mean Absolute Fragment Delta Mass [ppm]
01/14/2014 12:21 PM (8):Percolator: Features:
01/14/2014 12:21 PM (8):Percolator: enzyme=Trypsin
01/14/2014 12:21 PM (8):Percolator: Hyperparameters fdr=0.01, Cpos=0, Cneg=0, maxNiter=10
01/14/2014 12:21 PM (8):Percolator: Started Tue Jan 14 12:21:47 2014
01/14/2014 12:21 PM (8):Percolator: C:\Program Files\Thermo\Discoverer 1.4\Tools\Percolator\percolator.exe -X C:\ProgramData\Thermo\Discoverer 1.4\Scratch\1673be18-421e-4eb3-9044-64bec6a2781d\output.xml -Z C:\ProgramData\Thermo\Discoverer 1.4\Scratch\1673be18-421e-4eb3-9044-64bec6a2781d\input.xml
01/14/2014 12:21 PM (8):Percolator: Issued command:
01/14/2014 12:21 PM (8):Percolator: Department of Genome Sciences at the University of Washington.
01/14/2014 12:21 PM (8):Percolator: Written by Lukas Käll (lukall@u.washington.edu) in the
01/14/2014 12:21 PM (8):Percolator: Copyright (c) 2006-9 University of Washington. All rights reserved.
01/14/2014 12:21 PM (8):Percolator: Percolator version 2.04, Build Date Feb 1 2012 03:35:34
01/14/2014 12:21 PM (8):Percolator: Starting Percolator
01/14/2014 12:21 PM (8):Percolator: The input file contains 12113 peptides, 11068 decoy peptides and 32 features.
01/14/2014 12:21 PM (8):Percolator: Creating input file for Mascot (11) took 2 min 43 s.
01/14/2014 12:19 PM (8):Percolator: Start calculating features for peptides of Mascot (11)
01/14/2014 12:19 PM (11):Mascot: Used mascot server http://mascot0.icr.ac.uk/mascot/ with Mascot version 2.3.2
01/14/2014 12:19 PM (11):Mascot: Sending 10030 peptide hits (34449 peptides) to result file
01/14/2014 12:19 PM (11):Mascot: Sending 8251 decoy peptide hits (32129 peptides) to result file
01/14/2014 12:19 PM (11):Mascot: Reading decoy results
01/14/2014 12:19 PM (11):Mascot: Start translating results
01/14/2014 12:19 PM (11):Mascot: Start mapping modifications
01/14/2014 12:18 PM (11):Mascot: Received 1237 proteins from Mascot server
01/14/2014 12:18 PM (11):Mascot: Start mapping 1237 proteins
01/14/2014 12:18 PM (11):Mascot: Start parsing results
01/14/2014 12:18 PM (11):Mascot: Received Mascot result file (filename=../data/20140114/F051203.dat)
01/14/2014 12:18 PM (11):Mascot: Mascot Server completed
01/14/2014 12:14 PM (11):Mascot: Mascot result on server (filename=../data/20140114/F051203.dat)
01/14/2014 12:14 PM (11):Mascot: Start searching 22420 spectra
01/14/2014 12:14 PM (2):Scan Event Filter: 420 of 420 spectra pass this filter
01/14/2014 12:14 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:14 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:14 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:13 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:12 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:11 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:11 PM (11):Mascot: Use mascot server http://mascot0.icr.ac.uk/mascot/ with Mascot version 2.3.2
01/14/2014 12:11 PM (2):Scan Event Filter: 1000 of 1000 spectra pass this filter
01/14/2014 12:11 PM (1):Spectrum Selector: Reading from File 1 of 1:E:\Data\PR150\10993.raw (32738 spectra total)
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Common/Merged Quantification Method:
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General:
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Treat Quan Results as Replicates: False
Ratio Calculation:
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Show the Raw Quan Values = False
Minimum Quan Value Threshold: = 0
Replace Missing Quan Values With Minimum Intensity = False
Use Single-Peak Quan Channels = False
Apply Quan Value Corrections = True
Reject All Quan Values If Not All Quan Channels Are Present = False
Fold Change Threshold for Up-/Down-Regulation: = 2
Maximum Allowed Fold Change: = 100
Use Ratios Above Maximum Allowed Fold Change for Quantification = False
Percent Co-Isolation Excluding Peptides from Quantification: = 100
Protein Quantification:
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Use Only Unique Peptides
Experimental Bias:
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None
================================================================================
Search name: 10993
Search description: -
Search date: 01/14/2014 11:20:57
================================================================================
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Processing node 6: Reporter Ions Quantifier
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1. Quantification Method:
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Quantification Method: iTRAQ 4plex (Custom)
2. Peak Integration:
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Integration Tolerance: 20 ppm
Integration Method: Most Confident Centroid
3. Scan Event Filters:
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Mass Analyzer: FTMS
MS Order: MS2
Activation Type: HCD
Min. Collision Energy: 0
Max. Collision Energy: 100
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Quantification Method: iTRAQ 4plex
Method Description: Method for iTRAQ™ 4-plex mass tags by Applied Biosystems
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Quan Channels:
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Residue Modification:: iTRAQ4plex / +144.102 Da (K, Y)
N-Terminal Modification:: iTRAQ4plex / +144.102 Da (Any N-Terminus)
114: monoisotopic m/z = 114.11068 Da, average m/z = 114.17347 Da
115: monoisotopic m/z = 115.10771 Da, average m/z = 115.16688 Da
116: monoisotopic m/z = 116.11107 Da, average m/z = 116.15954 Da
117: monoisotopic m/z = 117.11442 Da, average m/z = 117.15219 Da
Purity Correction Factors in [%]:
-2 -1 0 1 2
114: 0.00 1.00 92.90 5.90 0.20
115: 0.00 2.00 92.30 5.60 0.10
116: 0.00 3.00 92.40 4.50 0.10
117: 0.10 4.00 92.40 3.50 0.00
Ratio Calculation:
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==> Minimum Quan Value Detected = 128.5
==> Minimum Quan Value Used = 128.5
Ratio Reporting:
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115/114
117/116 |
| instruments | LTQ Velos Orbitrap mass spectrometer (Thermo Fisher Scientific) |
| species | Human |
| massModifications | iTRAQ(114, 115, 116 and 117) |
