Proteins were identified from the tandem mass spectra extracted by Xcalibur version 4.0. MS/MS spectra were searched against the SwissProt Homo sapiens database using Mascot search engine (Matrix Science, London, UK; version 2.7.0). All searches included carbamidomethyl cysteine as a fixed modification and oxidized Lys, Pro, and Met; deamidated Asn and Gln; and acetylated N-term as variable modifications. Three missed tryptic cleavages were allowed. The MS1 precursor mass tolerance was set to 10 ppm and the MS2 tolerance was set to 0.05 Da. The search result was visualized by Scaffold v 5.0(Proteome Software, INC., Portland, OR). A 1% false discovery rate cutoff was applied at the peptide level. Only proteins with a minimum of two unique peptides above the cutoff were considered for further study. Raw mass spectrometry data will be made available upon publication and via request to the authors. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019 (link)) partner repository with the dataset identifier PXD026102 and 10.6019/PXD026102 and will be made publicly available upon acceptance of the manuscript.
Scaffold v 5
Manufactured by Proteome Software
Sourced in United States, Jamaica
Scaffold v 5.0 is a bioinformatics software application designed for the analysis and visualization of mass spectrometry data. It provides a comprehensive platform for the identification and quantification of proteins, peptides, and post-translational modifications. The software offers a user-friendly interface and a range of analytical tools to facilitate the interpretation of complex proteomic data.
Automatically generated - may contain errors
Lab products found in correlation
Mascot search engine, by Matrix Science (3 mentions)
Q exactive hf hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 rapid separation nanolc, by Thermo Fisher Scientific (1 mentions)
Picochip, by New Objective (1 mentions)
X tandem, by Proteome Software (1 mentions)
Prism 9, by GraphPad (1 mentions)
Clariostar, by BMG Labtech (1 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (1 mentions)
Microqtof qiii mass spectrometer, by Bruker (1 mentions)
6 protocols using scaffold v 5
1
Mass Spectrometry-based Protein Identification
2
Mass Spectrometry-based Protein Identification
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Proteins were identified from the tandem mass spectra extracted by Xcalibur version 4.0. MS/MS spectra were searched against the SwissProt Homo sapiens database using Mascot search engine (Matrix Science, London, UK; version 2.7.0). All searches included carbamidomethyl cysteine as a fixed modification and oxidized Lys, Pro, and Met; deamidated Asn and Gln; and acetylated N-term as variable modifications. Three missed tryptic cleavages were allowed. The MS1 precursor mass tolerance was set to 10 ppm and the MS2 tolerance was set to 0.05 Da. The search result was visualized by Scaffold v 5.0(Proteome Software, INC., Portland, OR). A 1% false discovery rate cutoff was applied at the peptide level. Only proteins with a minimum of two unique peptides above the cutoff were considered for further study. Raw mass spectrometry data will be made available upon publication and via request to the authors. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019 (link)) partner repository with the dataset identifier PXD026102 and 10.6019/PXD026102 and will be made publicly available upon acceptance of the manuscript.
3
Quantitative Proteomic Analysis of Peptide Samples
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The obtained peptides were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using a Dionex UltiMate 3000 Rapid Separation nanoLC and a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher). Samples were loaded onto a house-packed C18 column and separated with a 5 to 40% gradient of solvent (0.1% FA in ACN) for 120 min by an analytical column (PicoChip, New Objective, Inc.). MS/MS spectra were searched against the SwissProt Mus musculus database using Mascot search engine (Matrix Science; version 2.7.0.1). All searches included carbamidomethyl Cys as a fixed modification and oxidized Met; deamidated Asn and Gln; and acetylated N-term as variable modifications. The search result was visualized by Scaffold v 5.0.1 (Proteome Software, Inc.). A 1% false discovery rate of the protein with a minimum of two unique peptides was identified. Statistical analysis, specifically, a Fisher’s exact test with a Benjamini–Hochberg multiple test correction, was performed for comparison between 200-SNA and 150-SNA groups (n = 4 and 3 samples per group, respectively).
4
Proteomic Identification of Mouse Proteins
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MS/MS samples were analyzed on the Sequest Sorcerer platform (Sagen-N Research, San Jose, CA). Sequest was set up to interrogate the Mus musculus protein database (17,278 entries, UniProt, GCA_000001635.8 from Ensembl (http://www.uniprot.org/ )) that includes frequently observed contaminants, assuming trypsin is used. Sequest selected proteins with fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10.0 ppm. Cysteine carbamidomethyl and methionine oxidation were specified as fixed and variable modifications, respectively. Scaffold v5.0.1 (Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identities. Peptide identities were accepted if they had >94.0% probability of achieving <1.0% FDR based on the Peptide Prophet algorithm with Scaffold delta-mass correction. Protein identities were accepted if they had >99.0% probability of achieving <1.0% FDR and contained at least two identified peptides. Protein probabilities were assigned using the Protein Prophet algorithm v5.049 (link). Proteins with similar peptides that could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
5
M. haemolyticus Protein Identification
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The validation of peptide and protein identifications based on MS/MS was performed in a Sequest (Thermo Fisher Scientific; version IseNode in Proteome Discoverer 1.4.1.14) and X! Tandem (The GPM, thegpm.org; version X! Tandem Alanine, 2017.2.1.4 in Scaffold v5.1.2 Proteome Software Inc., Portland, Oregon) against the UniProtKB M. haemolityca database, 12,649 entries. If a peptide identification could be verified with a probability higher than 95.0%, it was deemed legitimate. Protein identifications with at least two identified peptides and a proven probability of greater than 99.0% were acceptable. We used the following search parameters: the digestion enzyme trypsin allows two missed cleavage; fragment ion mass tolerance of 0.60 Da; a parent ion tolerance of 20 PPM; carbamidomethylation of cysteine residues as fixed modification; oxidation of methionine residues deamidated of asparagine and glutamine as variable modification.
6
Quantitative Proteomic Analysis Workflows
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SDS–PAGE gels and on-cell assay data was collected using BioRad Gel Doc XR+ gel imager, BioRad ChemiDoc XRS+ gel imager and GE ImageQuant LAS 4000 gel imager. On-cell assay quantitative data were collected using the CLARIOstar plate reader (BMG labtech). MS data were collected using a Bruker microQTOF-QIII mass spectrometer. LC–MS/MS measurements were performed on an Orbitrap Eclipse TribridTM mass spectrometer (Thermo Fisher Scientific) equipped with an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) using a nanoEase M/Z column (HSS C18 T3, 100 Å, 1.8 μm; Waters). For SDS–PAGE analysis, we used Image Lab (v.6.1.0, build 7). For DNA sequence analysis, we used Staden 2.0.0b11-2016 and SnapGene. For graph generation, we used Microsoft Excel (v.2210, build 16.0.15726.20188) and Graphpad Prism 9. MS/MS data analysis was performed using Scaffold v.5.1.2 (Proteome Software Inc.). Protein structure images were produced using UCSF chimera 1.16. Routine liquid chromatography–MS analysis was carried out as previously described51 (link).
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