Scaffold v4.8.7 (Proteome Software Inc., Portland, OR, USA) was used to validate the MS/MS-based peptide and protein identifications, based on the Peptide Prophet algorithm [32 (link)] with Scaffold delta-mass correction and the Protein Prophet algorithm [33 (link)], respectively. Peptides had to be identified with at least 95% probability, and only those proteins identified with a minimum 95% probability (resulting in a protein FDR < 1%) using at least two unique peptides were analyzed further. Spectral counts were exported from Scaffold for all stable cell lines and formatted for SAINTexpress analysis [10 (link)] (available at https://reprint-apms.org ; accessed on 14 March 2021). Proteins with a SAINTexpress score ≥0.6 were incorporated into a network figure, highlighting the high confidence interactors scoring ≥0.6 and ≥0.95. Protein network mapping information was visualized using Cytoscape (v3.6.1).
Scaffold v4
Manufactured by Proteome Software
Sourced in United States
Scaffold v4.4.6 is a software application for analyzing and visualizing proteomic data. It provides a comprehensive platform for processing, organizing, and interpreting mass spectrometry-based proteomic data.
Automatically generated - may contain errors
Lab products found in correlation
Protein lynx global server version 2, by Waters Corporation (4 mentions)
Nanoacquity uplc system, by Waters Corporation (4 mentions)
Igepal ca 630, by Merck Group (3 mentions)
Dynabeads epoxy m270, by Thermo Fisher Scientific (3 mentions)
Xevo g2 qtof, by Waters Corporation (3 mentions)
Mascot, by Matrix Science (3 mentions)
Protease inhibitor mix, by Roche (2 mentions)
C18 waters trizaic nanotile, by Waters Corporation (2 mentions)
Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Mascot daemon, by Matrix Science (2 mentions)
Cytoscape v, by Cytoscape (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mix, by Merck Group (1 mentions)
Gfp trap a beads, by Proteintech (1 mentions)
Simplybluestain, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Nanoacquity, by Waters Corporation (1 mentions)
Sas statistical package v 9, by SAS Institute (1 mentions)
Proteome discoverer, by Thermo Fisher Scientific (1 mentions)
22 protocols using scaffold v4
1
Protein Identification and Interaction Analysis
2
Validating Protein Interactions by Mass Spectrometry
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Scaffold v4.8.7 (Proteome Software Inc., Portland, OR, USA) was used to validate the MS/MS-based peptide and protein identifications, based on the Peptide Prophet algorithm [92 (link)] with Scaffold delta-mass correction and the Protein Prophet algorithm [93 (link)], respectively. Peptides had to be identified with at least 95% probability, and only proteins identified with a minimum 95% probability (resulting in a protein FDR < 1%), using at least two unique peptides, were analyzed further. Spectral counts were exported from Scaffold for all four stable HEK293 cell lines and the HEK293 control and formatted for SAINTexpress analysis [15 (link)], a computational algorithm integrated into the REPRINT website (available at https://reprint-apms.org (accessed on 14 March 2021)). Proteins with a SAINTexpress score ≥0.6 were incorporated into a network figure, highlighting the high confidence interactors scoring ≥0.9 and ≥0.99. Network mapping information was exported from REPRINT and visualized with Cytoscape (v3.6.1).
3
Equine Proteome Identification Protocol
4
Protein Complex Purification in Maize
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Ten grams of wild‐type SAM maize tissue was used for each replicate, and nod‐1 alleles were used as a negative control. Membrane proteins were extracted using buffer A [50 mm Tris–HCl, pH 7.5, 150 mm NaCl, 2% IGEPAL‐CA‐630 (Sigma‐Aldrich, St. Louis, MO, USA) and 1 × protease inhibitor mix (Roche, Basel, Basel‐Stadt, Switzerland)]. Protein complex was purified using Dynabeads Epoxy M270 (Thermo Fisher Scientific) with Anti NOD antibody covalently coupled. After four washes with 1 × PBS + T, bound target proteins were eluted with a soft elution buffer [50 mm Tris–HCl, pH 7.5, 0.2% SDS, 0.1% Tween 20]. The complex was precipitated with acetone and then separated by SDS–PAGE. Western blot with specific antibodies confirmed the presence of the bait protein. Protein complex was in‐gel trypsin digested and analyzed by LC–MS/MS, using a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). For protein identification, Uniprot Zea mays database in the Mascot package was used. Results were exported into Scaffold v4.4.6 (Proteome Software, Portland, OR, USA).
5
Nanoflow-based Proteomics Analysis
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The mass spectrometry instrument used to analyze the samples was a Xevo G2 QTof coupled to a nanoAcquity UPLC system (Waters, Milford, MA, USA). Samples were loaded onto a C18 Waters Trizaic nanotile of 85 um × 100 mm; 1.7 μm (Waters, Milford, MA, USA). RAW files were processed using Protein Lynx Global Server (PLGS) version 2.5.3 (Waters, Milford, MA, USA). For viewing, PLGS search results were exported into Scaffold v4.4.6 (Proteome Software, Portland, OR, USA).
6
Affinity Purification of NOD Protein Complex
7
Mass Spectrometry Analysis of Protein Samples
8
Proteome Analysis by Nano-LC-MS/MS
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The mass spectrometry instrument used to analyze the samples was a Xevo G2 QTof coupled to a nanoAcquity UPLC system (Waters, Milford, MA). Samples were loaded onto a C18 Waters Trizaic nanotile of 85 um × 100 mm; 1.7 μm (Waters, Milford, MA). RAW files were processed using Protein Lynx Global Server (PLGS) version 2.5.3 (Waters, Milford, MA). For viewing, PLGS search results were exported into Scaffold v4.4.6 (Proteome Software Inc., Portland, OR).
9
Purification and Identification of Maize Protein Complexes
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Ten grams of wild type SAM maize tissue were used for each replicate and null alleles were used as a negative control. Membrane proteins were extracted using buffer A [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2% IGEPAL-CA-630 (Sigma) and 1× protease inhibitor mix (Roche)]. Protein complex was purified using Dynabeads Epoxy M270 (Thermo Fisher Scientific) with Anti NOD antibody covalently coupled. After four washes with 1XPBS+T, bound target proteins were eluted with a soft elution buffer [50 mM Tris-HCl, pH 7.5, 0.2% SDS, 0.1% Tween 20]. The complex was precipitated with acetone and then separated by SDS-PAGE. Western blot with specific antibodies confirmed the presence of the bait protein. Protein complex was in-gel trypsin digested and analyzed by LC-MS/MS, using a LTQ Orbitrap XL Mass spectrometer (Thermo Fisher Scientific). For protein identification, Uniprot Zea mays database in the Mascot package was used. results were exported into Scaffold v4.4.6 (Proteome Software Inc., Portland, OR).
10
Mascot Search and Scaffold Analysis
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Peak lists in the form of Mascot generic files (mgf files) were prepared from raw data using MS Convert (Proteowizard project) and sent to peptide match search on Mascot server v2.4.1 using Mascot Daemon (Matrix Science Ltd.).
Peak lists were searched against protein databases including typical proteomics contaminants such as keratins, etc. Tryptic peptides with up to two possible miscleavages and charge states +2, +3, +4 were allowed in the search. The following peptide modifications were included in the search: oxidized Methionine (variable), phosphorylated Serine/Threonine/Tyrosine (variable) and carbamidomethylated Cysteine (static). Data were searched with a monoisotopic precursor and fragment ion mass tolerance 10ppm and 0.6 Da respectively. Decoy database was used to validate peptide sequence matches. Mascot results were combined in Scaffold v4.4.0 (Proteome Software Inc) and exported to Excel (Microsoft Office) for further processing and comparisons.
In Scaffold, the peptide and protein identifications were accepted if probability of sequence match and protein inference exceeded 95.0% and 99% respectively. Protein probabilities were calculated in Scaffold by the Protein Prophet algorithm; proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony (Searle, 2010 (link)).
Peak lists were searched against protein databases including typical proteomics contaminants such as keratins, etc. Tryptic peptides with up to two possible miscleavages and charge states +2, +3, +4 were allowed in the search. The following peptide modifications were included in the search: oxidized Methionine (variable), phosphorylated Serine/Threonine/Tyrosine (variable) and carbamidomethylated Cysteine (static). Data were searched with a monoisotopic precursor and fragment ion mass tolerance 10ppm and 0.6 Da respectively. Decoy database was used to validate peptide sequence matches. Mascot results were combined in Scaffold v4.4.0 (Proteome Software Inc) and exported to Excel (Microsoft Office) for further processing and comparisons.
In Scaffold, the peptide and protein identifications were accepted if probability of sequence match and protein inference exceeded 95.0% and 99% respectively. Protein probabilities were calculated in Scaffold by the Protein Prophet algorithm; proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony (Searle, 2010 (link)).
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