Empty Flag constructs or Flag-NLRP3 were transfected into HEK293T cells for 24 h, and the cells were collected and resuspended in lysis buffer [50 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol, and 100 mM DTT (dithiothreitol)]. Extracts were immunoprecipitated with anti-Flag antibody and Protein A/G-Agarose beads and then dissolved in sample buffer. IP-enriched protein complexes were separated by SDS-PAGE, visualized by Coomassie blue staining, and then excised for in-gel digestion with trypsin. The peptides were extracted from gel bands and subjected to LC-MS/MS analysis. Tryptic peptides were separated on a C18 column and analyzed by an LTQ Orbitrap Velos mass spectrometer (Thermo). The resulting MS/MS data were processed using ProteinPilotTM software 4.5 (AB Sciex). The original MS/MS data were submitted to ProteinPilot (version 4.5, AB Sciex) for data analysis and searched against Homo sapiens in the UniProt database (http://www.uniprot.org/proteomes/UP000005640 ).
Proteinpilot version 4
Manufactured by AB Sciex
Sourced in United States
ProteinPilot is a software solution designed for protein identification and characterization. It utilizes advanced algorithms to analyze mass spectrometry data and provide reliable results. The software is intended to assist researchers in the identification and quantification of proteins within complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Triple tof 5600 system, by AB Sciex (2 mentions)
Proteinpilot software 4, by AB Sciex (1 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 nanolc system, by AB Sciex (1 mentions)
Triple tof 5600 lc ms system, by AB Sciex (1 mentions)
Mascot, by Matrix Science (1 mentions)
7 protocols using proteinpilot version 4
1
Flag-NLRP3 Immunoprecipitation and Mass Spectrometry
2
Proteomic Analysis of Primary Cortical Neurons
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Following reduction and alkylation, proteins collected from primary cortical neurons were trypsinized overnight at 37 °C. Peptides were dried and resuspended in mass-spectrometry-compatible buffer and then labeled with the 8-plex iTRAQ labeling reagent (Applied Biosystems, Foster City, CA, USA). Labeled samples were combined and analyzed with one-dimensional nanoLC-MS/MS (Dionex UltiMate 3000 nanoLC system coupled with AB Sciex TripleTOF 5600 system) for protein identification. The IPI human protein database (version 3.77) was searched using ProteinPilot (version 4.5, AB Sciex, Framingham, MA, USA) and the identified hits were analyzed using DAVID (http://david.abcc.ncifcrf.gov ; accessed on 11 January 2023) for gene ontology annotation [68 (link)]. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD036335 [69 (link)].
3
Automated Peptide Identification via LC-MS/MS
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A fully automated method was utilised. An information-dependent mass spectrometer experiment included 1 survey scan in MS1 followed by 50 dependent MS2 scans. The MS1 acquisition parameters were as follows: positive mode, mass range 300–1250 m/z, and signal accumulation time 250 milliseconds. Ions for MS2 analysis were selected based on an intensity above the threshold of 200 cps and a charge state from 2 to 5. MS2 acquisition parameters were as follows: resolution of quadrupole set to UNIT (0.7 Da), measurement mass range of 200–1800 m/z, and signal accumulation time of 50 milliseconds for each parent ion (using a 50-millisecond signal accumulation time). Analysed parent ions were sent to the dynamic exclusion list for 15 seconds to get an MS2 spectra at the chromatographic apex. Raw LC-MS/MS data were used for peptide identification with ProteinPilot (version 4.5) software (ABSciex) using the following parameters: Cys alkylation by iodoacetamide, trypsin digestion, and thorough ID search with detected protein threshold of 95.0% against the database.
4
Quantitative Proteomics by LC-MS/MS
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LC-MS/MS analysis was performed on a TripleTOF 5600+ system (SCIEX, MA, USA). Each sample was first loaded onto a trap column and then chromatographed using a 90-min gradient on an analytical column (75 μm × 15 cm, Magic C18 AQ 3-µm 120-Å). The eluted peptides were sprayed into mass spectrometer and scanned. MS data were acquired in DDA mode. MS1 spectra were collected in the range 350–1250 m/z for 250 ms. The 30 most intense precursors with charge state 2–5 were selected for fragmentation, and MS2 spectra were collected in the range 50–2000 m/z for 100 ms; precursor ions were excluded from reselection for 15 s.
Raw data were processed with ProteinPilot version 4.5 (SCIEX, MA, USA). MS and MS/MS spectra were searched against SwissProt human database using Paragon algorithm65 (link). The database search was performed with the following parameters: iTRAQ 8plex (peptide labelled) for Sample type according to the experiments; Iodoacetamide for Alkylation; Trypsin for Digestion; Phosphorylation emphasis for Special factors; Thorough ID for Search effort. ProteinPilot calculates a percentage of confidence that reflects the probability that the hit is a false positive. Only high-quality peptide assignments with confidence >95% were considered in this study.
Raw data were processed with ProteinPilot version 4.5 (SCIEX, MA, USA). MS and MS/MS spectra were searched against SwissProt human database using Paragon algorithm65 (link). The database search was performed with the following parameters: iTRAQ 8plex (peptide labelled) for Sample type according to the experiments; Iodoacetamide for Alkylation; Trypsin for Digestion; Phosphorylation emphasis for Special factors; Thorough ID for Search effort. ProteinPilot calculates a percentage of confidence that reflects the probability that the hit is a false positive. Only high-quality peptide assignments with confidence >95% were considered in this study.
5
Comprehensive MS-based Protein Identification
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MS analyses were carried out by GeneChem. Briefly, gel strips were obtained through the electrophoresis of magnetic immune complexes from co‐IP assays. Subsequently, protein gel strips underwent a series of sequential processes, including decolorization, alkylation, enzymatic hydrolysis, extraction, and desalination to yield peptide samples. These peptide samples were subjected to the TOF 5600 LC/MS system (AB SCIEX, Framingham, MA, USA). Original MS/MS files were then submitted to ProteinPilot (Version 4.5, SCIEX, Redwood, CA, USA) for comprehensive data analysis. For protein identification, the Paragon algorithm was utilized to search the Uniprot database (https://www.uniprot.org/ ). Peptides with an unused score > 1.3 (credibility exceeding 95%) were acknowledged as credible peptides.
6
Mass Spectrometry-based Protein Identification
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The MS analysis was performed by the GeneCreate Biological Engineering Co., Ltd (Wuhan, Hubei, China). Briefly, the magnetic immune complexes from co‐IP assays were electrophoresed to obtain gel strips. Then, protein gel strips were sequentially decolorized, alkylated, enzymatically hydrolyzed, extracted, and desalted into peptide samples. Then the peptide samples were analyzed by the Triple TOF 5600 + LC/MS system (AB SCIEX, Framingham, MA, USA). Finally, the original MS/MS files from the mass spectrometer were submitted to ProteinPilot (Version 4.5, SCIEX, Redwood, CA, USA) for data analysis. For protein identification, the Paragon algorithm in ProteinPilot was used to search the Uniprot database (https://www.uniprot.org/ ). Peptides with an unused score > 1.3 (a credibility of more than 95%) were considered credible peptides, and proteins containing at least one unique peptide are retained.
7
Leishmania Infantum Protein Identification
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MGF peak list files were created using Protein Pilot version 4.5 software (Sciex). MGF sample files were then analyzed using Mascot (Matrix Science, London, UK; version 2.5.1). Mascot was searched with a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 1.0 Da. Scaffold (version Scaffold_4.8.4, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications based on the Leishmania Infantum TriTrypDB (version 9.0 released April 2016, 8,589 entries). Peptide identifications were accepted if they could be established at greater than 5.0% probability to achieve an FDR less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve a FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm79 (link). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
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