All MS/MS peak lists (MGF files) were generated using ProteinPilot (AB Sciex, Version 4.5) with the Paragon algorithm. MGF peak list files were created using Protein Pilot version 4.5 software (ABSciex, Concord, Ontario, Canada) utilizing the Paragon and Progroup algorithms (Shilov). MGF sample files were then analyzed using Mascot (Matrix Science, London, UK; version 2.4.0). Mascot was searched with a fragment ion mass tolerance of 0.100 Da and a parent ion tolerance of 0.100 Da. Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Glu->pyro-Glu of the n-terminus, gln->pyro-Glu of the n-terminus, deamidation of asparagine and glutamine, and oxidation of methionine were specified in Mascot as variable modifications.
Proteinpilot
Manufactured by AB Sciex
Sourced in United States, Canada, Germany, United Kingdom
ProteinPilot is a software application developed by AB Sciex for protein identification and quantification. It provides a comprehensive solution for analyzing mass spectrometry data to identify and characterize proteins in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Peakview, by AB Sciex (7 mentions)
Mascot, by Matrix Science (6 mentions)
Formic acid, by Thermo Fisher Scientific (6 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (5 mentions)
Paragon algorithm, by AB Sciex (2 mentions)
Proteinpilot software v 4, by AB Sciex (2 mentions)
Up000000803, by UniProt (2 mentions)
5800 maldi tof tof, by AB Sciex (2 mentions)
Maldi tof tof 5800 system, by AB Sciex (2 mentions)
2d silver stain 2, by Cosmo Bio (2 mentions)
Mascot 2, by Matrix Science (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Acclaim pepmap rslc c18 column, by Thermo Fisher Scientific (2 mentions)
Tripletof 6600 ms, by AB Sciex (2 mentions)
Formic acid, by Merck Group (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Spectronaut 14, by Biognosys (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 nano lc hplc system, by Thermo Fisher Scientific (1 mentions)
Triple tof 5600 plus system, by AB Sciex (1 mentions)
83 protocols using proteinpilot
1
MS/MS Data Analysis Protocol
2
Comprehensive Brain Peptide Profiling
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This experiment was performed as previously described25 with some modifications in Triple Quad 6500 liquid chromatography–tandem mass spectrometry (AB SCIEX, Concord, Canada). Briefly, a C18 analytical column was prepared (0.075×150 mm, 3 μm, 120 A), and 2 mg of brain peptides was subjected to the EMS‐EPI (Enhanced Mass Spectrum‐Enhanced Product Ion) mode for identification. Then, the EMS‐EPI data were searched with ProteinPilot (AB SCIEX). Transitions for all identified peptides were designed by importing the ProteinPilot results files to MRMPilot (Skyline software; AB SCIEX) with the peptide selection criteria including a unique peptide for a protein and no missed cleavage. Finally, the 67 designed peptides (3 proteins with 5 peptides, 3 proteins with 4 peptides, 5 proteins with 3 peptides, 5 proteins with 2 peptides, and 15 proteins with 1 peptide) and 378 transitions were used to survey the protein digests from each brain using the multiple reaction monitoring (MRM) mode with 3 injection repeats. For MRM confirmation, the raw MRM data were processed using Skyline 3.5.9.10130.
3
Protein Identification and SWATH Peak Extraction
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Protein identifications from 2D-IDA data were performed with ProteinPilot (v5.0, Sciex) using the Paragon algorithm in thorough mode. The search parameters were as follows: sample type: identification; cys alkylation: iodoacetamide; digestion: trypsin + Lys C; instrument: TripleTOF 6600; special factors: none; species: Homo sapiens; ID focus: biological modifications. The database used was obtained from SwissProt (20,386 entries, August 2018). A reversed-decoy database search strategy was used with ProteinPilot, with the calculated protein FDR equalling 1%. The ProteinPilot group file from the 2D-IDA search result was imported into PeakView (v2.2; Sciex) and used as a local peptide assay library. This library contained 3208 identified proteins (S1 Table in S1 File ). SWATH peaks were then extracted using PeakView. Shared and modified peptides were excluded. Peak extraction parameters were set as the following: 100 peptides per protein, 6 transition ions per peptide, peptide confidence threshold 99%, FDR extraction threshold 1%, Extract Ion Chromatogram retention time window 5 min and mass tolerance 75 ppm. The extracted transition ion peak areas, peptide peak areas and protein peak areas were exported for further statistical analysis.
4
Mass Spectrometry-based Protein Identification
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The original MS/MS data of the mass spectrometer were transferred to the ProteinPilot (ProteinPilot-software">https://sciex.com.cn/products/software/ProteinPilot-software , accessed on 23 October 2021, version 4.5, SCIEX, Redwood City, CA, USA). For protein identification, the Paragon algorithm in ProteinPilot was used to search the uniprot-Bos_mutus-uniprot-organism_72004.fasta database. The parameters were set as follows: the instrument was TripleTOF 5600, and the cysteine was modified with iodoacetamide; the biological modification was selected as the ID focus. For identifying the protein certain filtering criteria were selected, and peptides with an unused score >1.3 (with a confidence level of 95% or more) were considered credible peptides, and the proteins containing at least 1 unique peptide were retained. The functional enrichment analysis of interacting genes with TSSK4 was performed using the g: Profile online website (https://biit.cs.ut.ee/gprofiler/gost , accessed on 13 December 2021) and KOBAS online website (http://kobas.cbi.pku.edu.cn/ , accessed on 13 December 2021).
5
Proteomic Identification of Annelida Proteins
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The protein database searches were conducted in ProteinPilot (software ver 4.5, AB SCIEX) using the Paragon algorithm against the Uniprot databases (ver. 8.11.2017, Annelida, Lophotrochozoa) with an automated false discovery rate (FDR). Search parameters: instrument TripleTOF 5600, alkylation of the cysteine residue by iodoacetamide, trypsin/chymotrypsin/Lys-C enzyme digestion, ID focus on biological modifications, search effort “thorough ID” and detected protein threshold [Conf] > 10%, and peptide confidence ≥ 95% and peptide FDR < 1% for identification of peptides.
6
Proteomic Profiling of Differentially Expressed Proteins
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Raw data from LC-MS/MS in the form of wiff files were analyzed using the ProteinPilot (AB SCIEX; Version: 4.5) for protein identification and quantitation, which was searched against the UniProt proteome dataset (species: rat; downloaded on May 31, 2017) that containing 36,663 entries. Unique peptides were characterized using an FDR (false discovery rate) of <0.05 using Benjamini-Hochberg method. Significantly differentially expressed proteins were defined by the combination of fold change (>1.5) and p value (<0.05). The R package “pheatmap” was used to perform the hierarchical clustering of differentially expressed proteins. Functional categorization of differentially expressed proteins based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases were done using the DAVID (Database for Annotation, Visualization and Integrated Discovery) website (https://david.ncifcrf.gov ).
7
CaMKK2 Autophosphorylation Analysis
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In order to analyse Thr85 autophosphorylation in the G87R mutant, 1 μg of WT CaMKK2 or G87R mutant was incubated with 5 mM MgCl2, 50 μM CaCl2 and 1 μM CaM in the presence and absence of 200 μM ATP for 40 min, then digested with trypsin and analyzed by reversed-phase HPLC-ESI-MS/MS using an UltiMate 3000 Nano LC HPLC system (Dionex) directly connected to a Triple-TOF 5600 mass spectrometer (AB SCIEX) in direct injection mode. Peptide mixtures were resolved on an analytical nanocapillary HPLC column (100 μm i.d. × 15 cm) packed with C18 Acclaim PepMap100 (3 μm particle size, 100 Å pore size) using a 1–75% elution gradient of 98% acetonitrile/2% of 0.1% formic acid (v/v) in water at a flow rate of 250 nl/min. Mass spectrometric data were analyzed using the database search engine ProteinPilot and the Paragon algorithm (AB Sciex).
8
Protein Identification and Quantification Protocol
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For protein identification and quantification, raw MS/MS data were submitted to Paragon in ProteinPilot (version 3, AB Sciex) and searched against a local CAC sequence database (a concatenation of NCBI references NC_003030.1 and NC_001988). Search parameters were the same as previously specified [75 (link)]. Bias correction and background correction were performed through ProteinPilot. Protein identifications were based on 95 % confidence or above, and only proteins with at least one peptide with 95 % confidence were included in the quantified protein list. For evaluation of the protein identification false discovery rate (FDR), the MS/MS data were submitted to a decoy database and FDR was calculated from the ratio of the number of hits from the decoy database to the number of hits from normal and decoy database (Additional file 2 : Proteomic data).
Protein expression levels under BuOH or BA stress were compared to the controls under no stress with the same reference using significance analysis of microarrays (SAM analysis [76 (link)]) with MeV v4.8 [77 (link)] as reported [75 (link)]. The delta values were set for an FDR of 5 % as cutoff.
Protein expression levels under BuOH or BA stress were compared to the controls under no stress with the same reference using significance analysis of microarrays (SAM analysis [76 (link)]) with MeV v4.8 [77 (link)] as reported [75 (link)]. The delta values were set for an FDR of 5 % as cutoff.
9
Mouse Protein Identification by Mass Spectrometry
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The mass spectrometry files were searched using Protein Pilot (AB SCIEX, Concord, Canada) and Mascot (Matrix Science Inc., Boston, USA). Samples were input in the Protein Pilot software v. 4.2 (AB SCIEX, Concord, Canada), which employs the Paragon algorithm, with the following parameters: cysteine alkylation, digestion by trypsin, no special factors and false discovery rate (FDR) at 1%. The UniProt Swiss-Prot reviewed database containing mouse proteins (version 20july15, containing 23,304 sequence entries). The Mascot search was performed on Mascot v. 2.4, the digestion enzyme selected was trypsin, with 2 missed cleavages and a search tolerance of 50 ppm was specified for the peptide mass tolerance, and 0.1 Da for the MS/MS tolerance. The charges of the peptides to search for were set to 2+, 3+ and 4+, and the search was set on monoisotopic mass. The instrument was set to ESI-QUAD-TOF (electrospray ionization quadrupole time-of-flight) and the following modifications were specified for the search: carbamidomethyl cysteines as fixed modification and oxidized methionine as variable modification.
10
Mass Spectrometry Identification of HBsAg-bound Proteins
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The SDS-PAGE gels in far-western blotting corresponding to the HBsAg-bound proteins were subjected to mass spectrometry analysis using a TripleTOF 5600-plus system (AB Sciex, Foster City, CA, USA) coupled with an Ekspert NanoLC 425 (AB Sciex). ProteinPilot (AB Sciex) was used to process the acquired time-of-flight mass spectra data for peptide and protein identification.
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