Database search was performed with ProteinPilot 4.5 software (Sciex, v.4.5 AB, Framingham, MA, USA) using the Paragon algorithm against the UNIPROT Dickeya solani database with an automated false discovery rate, and standard parameters [79 (link),80 (link)]. Next, a spectral library was created with the group file data processing in PeakView v. 2.2 (Sciex), with parameters as described in detail by Lewandowska [79 (link)]. Files from SWATH experiments for each sample were downloaded to PeakView (Sciex, v.2.2, Framingham, MA, USA) software and processed with the previously established library. Resulting data were exported to the .xml file and exported to Marker View software. All data were normalized using total area sums (TAS) approach, grouped as wild type and tested samples and t-test was performed. Samples were compared to each other, coefficient of variation (CV%) was calculated, and proteins with a p-value lower than 0.05 with fold change 2 were considered as differentially expressed in examined samples. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [81 (link)] partner repository with the dataset identifier PXD018297.
Proteinpilot 4
Manufactured by AB Sciex
Sourced in United States
ProteinPilot 4.5 software is a data analysis tool designed for the identification and quantification of proteins from mass spectrometry data. It provides an automated workflow for the processing and interpretation of proteomics data.
Automatically generated - may contain errors
Lab products found in correlation
Tripletof 5600 mass spectrometer, by AB Sciex (10 mentions)
Tripletof 5600, by AB Sciex (5 mentions)
Silver staining kit, by Beyotime (5 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (3 mentions)
Nanolc ultra 2d, by AB Sciex (3 mentions)
Coomassie blue r250, by Solarbio (3 mentions)
Peakview, by AB Sciex (2 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (2 mentions)
Protein a g magnetic beads, by Merck Group (2 mentions)
Protease and protein phosphatase inhibitors, by Roche (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Nanospray 3 source, by AB Sciex (2 mentions)
Tripletof 5600 ms, by AB Sciex (2 mentions)
Tripletof 5600 hybrid mass spectrometer, by AB Sciex (2 mentions)
Peak viewer, by AB Sciex (2 mentions)
Marker viewer, by AB Sciex (2 mentions)
4800 maldi tof tof, by AB Sciex (1 mentions)
Peakview v 2, by AB Sciex (1 mentions)
Paragon algorithm, by AB Sciex (1 mentions)
Pepclean, by Thermo Fisher Scientific (1 mentions)
70 protocols using proteinpilot 4
1
Quantitative Proteomics Workflow for Dickeya solani
2
MALDI-TOF/TOF Protein Identification Protocol
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MS data were acquired on a 4800 MALDI TOF/TOF (AB Sciex, Framingham, MA) at a laser repetition rate of 200 Hz with 600 laser shots/spectrum (50 laser shots/sub-spectrum). MS/MS data were acquired at 200 Hz in 2 kV MS/MS mode with 2250 laser shots/spectrum (50 laser shots/sub-spectrum) with the following TOF/TOF Series Explorer Stop Conditions: maximum shots per spectrum 2,250, minimum shots per spectrum 800, number of MS/MS fragments 8, with a signal/noise ratio of each fragment 75. Typically, the top 30 strongest peaks were selected for MS/MS analysis. Raw data were transformed to mascot generic format using ProteinPilot 4.5 (Sciex).
3
Identification of Prevalent Peptide Modifications
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We conducted a PTM analysis with the purpose of identifying the most commonly single observed peptide modifications, based on the data from the colon biopsies. Mascot generic format files were generated from the raw data-files in Proteome Discoverer 1.4 (Thermo Scientific, Waltham, USA). The Mascot generic format files were searched individually using ProteinPilot 4.5 (Rev. 1656, Paragon algorithm 4.5.0.0 [33] (link)) (SCIEX, Framingham, USA) against the Uniprot Homo sapiensreference proteome (UP000005640, last modified 2015-01-16, protein count 68,015). The files were searched in ⿿thorough⿿ mode with a focus on biological modifications to include 303 different PTMs. To give a representation of the global PTM distribution the search result was analyzed using ProteinPilot Descriptive Statistics Template version 3.001 (SCIEX) according to manufacturers⿿ instructions. The statistics template included the first 20,000 peptide spectral matches (PSMs) resulting in the identification of peptides with <1% local peptide false discovery rate (FDR). A stricter filtering setting than the standard <5% local peptide FDR was applied for included peptides, to ensure that only high confidence data was included in the analysis.
4
Protein Identification and Quantification using TripleTOF 5600
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ProteinPilot™ 4.5 (SCIEX) was applied to protein identification and iTRAQ quantification, using SwissProt (2013_09, total 540958 sequences) as the database. The search parameter settings were: Cysteine alkylation with MMTS; Trypsin Digestion; TripleTOF 5600; Biological modifications. The identified proteins were grouped using the ProGroup algorithm to eliminate the redundancy. A decoy database search strategy was applied to determine the false discovery rate (FDR). Unused score >1.3 (corresponding to a protein confidence interval >95%) was applied as the cutoff threshold for protein identification, with a false discovery rate (FDR) of 0.33%.
5
ProteinPilot Analysis of Plasmodium Proteome
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The detailed methods of ProteinPilot analysis have been illustrated previously60 (link). The proteins were identified with ProteinPilot 4.5 (SCIEX) that uses a Paragon algorithm to carry out database searches. PlasmoDB (v13, P. falciparum 3D7) was used as the database, while the search parameters used were as follows: cysteine alkylation with MMTS; trypsin digestion; TripleTOF 5600; and biological modifications. The identified proteins were grouped using the ProGroup algorithm in the software to remove any redundancy. To determine the false-discovery rate (FDR) for protein identification, a decoy database search strategy was utilized, of which a corresponding randomized database was produced using the Proteomics System Performance Evaluation Pipeline feature in the ProteinPilot 4.5 software. An unused score ≥1.3 was used as the cutoff threshold for protein identification, after which the FDR equals to 0.
6
Protein Identification by Tandem MS
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The tandem mass spectrometry data were processed to provide protein identifications using an in-house Protein Pilot 4.5 search engine (Sciex) using the Homo sapiens Sprot protein database and using a trypsin digestion parameter. Proteins of significance were accepted on the criteria of having at least two peptides detected with a confidence score of 95% or greater.
7
Quantitative Analysis of Sialolith Proteins
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For quantitative analysis of four sialoliths samples, a spectral library was created with the group file data processing in PeakView v. 2.2 (SCIEX), with settings described in detail by Lewandowska [57 (link)]. Joint search for library generation included all S2–S5 samples and pooled sample. For database search, ProteinPilot 4.5 software (Sciex) was used. It is based on the Paragon algorithm against the dedicated SwisProt Homo sapiens database (4 December 2020) with an automated false discovery rate. All files from SWATH experiments for sialoliths were downloaded to PeakView software and were processed with the previously established library. The resulting data were exported to an .xml file and exported to Marker View software. All data were normalized using the total area sums (TAS) approach. T-tests were conducted to find significant differences between protein concentration in tested samples. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier [58 ]. Cytoscape 3.8.0 [59 (link)] and STRING 11.0 [60 (link)] were used for the interactome network visualization.
8
Proteomic Identification and Quantification
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Raw spectra from WIFF files containing MS and MS/MS data were analyzed using Protein Pilot 4.5 software using Paragon algorithm (Sciex). Protein identification parameter of carbamidomethylation of cysteine was selected. All LC–MS samples were searched against the curated UniProt's human proteome release (2017_02) containing 42 147 protein entries for unique canonical sequence and splice isoforms. Proteins and peptides identified at >95% confidence (unused score > 1.3) were used for subsequent comparative quantitative analysis. Gene ontology analysis for molecular function of the mass spectrometry results from the pull-down assay was performed using PANTHER web services (19 ,41 (link),42 ) using the GO Ontology database, released 28 February 2017.
9
Disulfide Peptide Identification in Mtb Pck
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To identify native disulfide-containing peptides, wt MTb Pck was digested by pepsin in acidic pH [11 (link)]. Briefly, 10 μg of protein in 20 mM Tris buffer, 150 mM NaCl, pH 8, were mixed with 400 μl of 0.5% trifluoroacetic acid and 100 μl of 8 M urea. Pepsin dissolved in water was added in an pepsin:protein ratio of 1:50. The digestion was performed for 2 h at room temperature. The resulting peptides were desalted on a C18 SPE column (PepClean, Thermo). During LC-MS/MS, the peptides were separated with a nano LC system (Ultimate 3000 RSLC nano, Dionex) on an AcclaimPepMap C18 column (75 um Internal Diameter, 250 mm length) using a 70 min elution gradient of acetonitrile in 0.1% formic acid. MS/MS data were acquired on a tandem mass spectrometer (TripleTOF 5600, Sciex) in a data-dependent manner selecting up to 15 precursor ions for fragmentation in each cycle. The resulting raw data were searched against a database consisting of mycobacterial proteins as well as proteins from E. coli and common contaminants in Protein Pilot 4.5 (Sciex). Ions suspected of containing disulfides were subjected to manual curation to confirm the position of the disulfide bridge within the peptide molecule.
10
Proteomics Analysis of Voltage-Gated Sodium Channels
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Identification of the proteins was done using Protein Pilot® 4.5 (Sciex, Redwood City, USA) and all data dependent analysis (DDA) runs MS/MS spectra were identified against respective Nav channel protein data retrieved from UniprotKB/SwissProt library. FDR 1% and 99% peptide confidence level was used in the library creation and only distinctive peptides were used in the identification. Mass accuracy was set to 5 ppm for each peptide.
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