Mascot

Manufactured by Matrix Science

Sourced in United Kingdom, United States, Germany, Canada

Mascot is a versatile lab equipment designed for efficient sample preparation and analysis. It features a compact and durable construction, enabling reliable performance in various laboratory settings.

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Lab products found in correlation

941 protocols using mascot

Proteomic Analysis of Acetylated Proteins

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Raw data were processed with Raw2MSN [34 (link)], and Mascot generic files generated were analyzed using Mascot (Matrix Science Ltd, London, UK, version 2.4.1), querying both: SwissProt database (release 2014_01, restricted to Homo sapiens, 20 273 entries) and an in‐house database containing common proteomic contaminants and main isoforms of human p53. Mascot was searched assuming the digestion enzyme trypsin allowing for two miscleavages with a fragment ion mass tolerance of 0.5 Da and a parent ion tolerance of 10 p.p.m. Cysteine carbamidomethylation was specified in Mascot as a fixed modification. Acetylation of protein N termini and lysine residues, oxidation of methionine moieties, and phosphorylation of serine and threonine residues were specified in Mascot as variable modifications. scaffold (version 4.3.2; Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS‐based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm, resulting in a peptide false discovery rate of 0.02% [35 (link)]. Only acetylated peptides still present after filtering were used to build the library used in PRM experiments.

Marx C., Sonnemann J., Beyer M., Maddocks O.D., Lilla S., Hauzenberger I., Piée‐Staffa A., Siniuk K., Nunna S., Marx‐Blümel L., Westermann M., Wagner T., Meyer F.B., Thierbach R., Mullins C.S., Kdimati S., Linnebacher M., Neri F., Heinzel T., Wang Z, & Krämer O.H. (2021). Mechanistic insights into p53‐regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells. Molecular Oncology, 15(12), 3404-3429.

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Mass Spectrometry Peptide Identification

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MS/MS data were searched against the NCBInr human database with Mascot (v2.3.2, Matrix Science) using Mascot Distiller (2.3.2.0) as the data input filter. Search parameters were set as follows: enzyme, trypsin; precursor ion mass tolerance, 10 ppm; fragment ion mass tolerance, 0.3Da; maximum missed cleavages allowed 2; carbamidomethyl of cysteine residues for fixed modification; oxidation of methionine and addition of 156. 07864 Da on lysine or N-terminal (end-capping modification) for variable modification. The criteria used to filter results included 1% false positive threshold and expect value of less than 0.05 for significant peptide matches. The expect score was calculated using the homology threshold or the significance threshold as per a standard Mascot protein family report.

Huang B.X., Chen H., Joo Y., Kwon H.S., Fu C., Spector A.A, & Kim H.Y. (2023). Interaction between GPR110 (ADGRF1) and tight junction protein occludin implicated in blood-brain barrier permeability. iScience, 26(4), 106550.

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Mass Spectrometry-based Protein Quantification

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Acquired MS spectra were imported into Progenesis software (version 2.5, Nonlinear Dynamics, Waters, Oslo, Norway) and analyzed as previously described [76 (link),77 (link)]. After alignment, peak picking, exclusion of features with charge state of 1 and >7 and normalization, spectra were exported as Mascot Generic files and searched against a database containing all entries of Mycobacterium avium subspecies paratuberculosis from NCBI Protein database combined with the Ensembl bovine database (version 80) with Mascot (Matrix Science, Version 2.5.1). Search parameters used were 10 ppm peptide mass tolerance, 20 mmu fragment mass tolerance, one missed cleavage allowed, carbamidomethylation set as fixed modification, and methionine oxidation and deamidation of asparagine and glutamine as variable modifications. Mascot integrated decoy database search was set to a false discovery rate (FDR) of 1% when searching was performed on the concatenated mgf files with a percolator ion score cut-off of 13 and an appropriate significance threshold p. Peptide assignment was reimported to Progenesis Software. All unique peptides allocated to a protein were considered for quantification.

Kleinwort K.J., Hobmaier B.F., Mayer R., Hölzel C., Degroote R.L., Märtlbauer E., Hauck S.M, & Deeg C.A. (2021). Mycobacterium avium subsp. paratuberculosis Proteome Changes Profoundly in Milk. Metabolites, 11(8), 549.

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Soybean Protein Mass Spectrometry

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Total protein was tryptic digested prior to being subjected to LC-MS/MS using a Orbitrap-Velos instrument (Thermo Fisher, Waltham, MA, United States). Tandem mass spectra were extracted; charge state deconvolution and deisotoping were not performed. MS/MS samples were analyzed using Mascot (Matrix Science, London, United Kingdom; Mascot in Proteome Discoverer 2.4.0.305). Mascot was set up to search the Refseq database Glycine_max (86,460 entries), assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine (+57 on C) was specified in Mascot as a fixed modification. Oxidation of methionine (+16 on M) was specified in Mascot as a variable modification.

Ilangumaran G., Subramanian S, & Smith D.L. (2022). Soybean Leaf Proteomic Profile Influenced by Rhizobacteria Under Optimal and Salt Stress Conditions. Frontiers in Plant Science, 13, 809906.

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Proteomic Analysis of Pathogenic Bacteria

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Data were analyzed using Mascot (Matrix Science) with the following parameters: Enzyme: Trypsin/P; Database: UniProt F. tularensis SCHU S4 or UniProt B. pseudomallei strain 1026b (forward and reverse appended with common contaminants and mouse IgG sequences); Fixed modification: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q), Deamidation (N/Q); Mass values: Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2; Mascot DAT files were parsed into Scaffold Proteome Software for validation, filtering and to create a non-redundant list per sample. Data were filtered using 1% protein and peptide FDR and requiring at least one unique peptide per protein.

D’haeseleer P., Collette N.M., Lao V., Segelke B.W., Branda S.S, & Franco M. (2021). Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei. Frontiers in Immunology, 12, 716676.

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Progenesis QI for Label-free Proteomics

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Progenesis QI for proteomics software (version 2.4; Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK) was used to perform ion intensity–based label-free quantification as previously described (54 (link)). In an automated format, .RAW files were imported and converted into two-dimensional maps (y axis, time and x axis, m/z), followed by selection of a reference run for alignment purposes. An aggregate dataset containing all peak information from all samples was created from the aligned runs, which was then further narrowed down by selecting only +2, +3, and +4 charged ions for further analysis. The samples were then grouped, and a peak list of fragment ion spectra from only the top eight most intense precursors of a feature was exported to a Mascot generic file (.MGF) format and searched using Mascot (version 2.4; Matrix Science, London, UK) with the same search variables as described above. The resulting Mascot.XML file was then imported into Progenesis, allowing for peptide/protein assignment, whereas peptides with a Mascot ion score of <25 were not considered for further analysis. Protein quantification was performed using only nonconflicting peptides, and precursor ion abundance values were normalized in a run to those in a reference run (not necessarily the same as the alignment reference run).

Duron D.I., Lei W., Barker N.K., Stine C., Mishra S., Blagg B.S., Langlais P.R, & Streicher J.M. (2020). Inhibition of Hsp90 in the spinal cord enhances the antinociceptive effects of morphine by activating an ERK-RSK pathway. Science signaling, 13(630), eaaz1854.

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Proteomics Profiling by LC-MS/MS

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LC-MS/MS analyses were performed on a linear ion trap (LTQ) tandem mass spectrometer (LTQ-FT LC/MS/MS, Thermo Electron). The Mascot search engine (Matrix Science) was used for protein identification according to a curated protein database (IPI human, International Protein Index, compiled by the European Bioinformatics Institute). Peptide mass tolerance and fragment mass tolerance were set at 100 ppm and 0.25 Da, respectively. Proteins were scored using a probability-based MOWSE (for MOlecular Weight SEarch) algorithm, and the Mascot scores were reported in the form of −10 × log(P), where P is the probability that the observed match is a random event. The acquired data was searched against the International Protein Index (IPI) human protein sequence database by using the automated database-searching program, Mascot (Matrix Science). Spectra were searched with a mass tolerance of 15 ppm for MS data and 0.8 Da for MS/MS data. Up to two missed trypsin cleavages was allowed. Carbamidomethyl cysteine was set as a fixed modification, and oxidized methionine and deamidation was set as variable modifications. All identified proteins were summarized and exported as spread sheet in .xlsx file format.

Chi L.H., Chang W.M., Chang Y.C., Chan Y.C., Tai C.C., Leung K.W., Chen C.L., Wu A.T., Lai T.C., Li Y.C, & Hsiao M. (2017). Global Proteomics-based Identification and Validation of Thymosin Beta-4 X-Linked as a Prognostic Marker for Head and Neck Squamous Cell Carcinoma. Scientific Reports, 7, 9031.

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Mascot-Based Proteomics Data Analysis

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Mascot generic format peak list files were created using Protein Pilot, version 4.5 software (Sciex) for the data obtained with the 5600+ triple TOF and with Proteome Discoverer 2.3 software (Thermo) for the Orbitrap data. The Mascot generic format sample files were then analyzed using Mascot (Matrix Science; version 2.5.1). Mascot was set up to search a contaminant database and UniProtKB Homo sapiens database assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.60 Da (Orbitrap) or 0.1 Da (5600+) and a parent ion tolerance of 10.0 ppm (Orbitrap) and 0.1 Da (5600+). Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Deamidation of asparagine and glutamine and oxidation of methionine were specified in Mascot as variable modifications. Two missed cleavages were allowed.

Devoucoux M., Roques C., Lachance C., Lashgari A., Joly-Beauparlant C., Jacquet K., Alerasool N., Prudente A., Taipale M., Droit A., Lambert J.P., Hussein S.M, & Côté J. (2022). MRG Proteins Are Shared by Multiple Protein Complexes With Distinct Functions. Molecular & Cellular Proteomics : MCP, 21(7), 100253.

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Quantitative Proteomics of Mycobacterium avium

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Acquired MS spectra were imported into Progenesis software (Version 2.5 Nonlinear Dynamics, Waters) and analyzed as previously described (Hauck et al., 2012 (link); Hauck et al., 2017 (link)). After alignment, peak picking, exclusion of features with charge state of 1 and >7 and normalization, spectra were exported as Mascot Generic files and searched against a database containing all entries of Mycobacterium avium subspecies paratuberculosis from NCBI Protein database combined with the Ensembl bovine database (Version 80) with Mascot (Matrix Science, Version 2.5.1). Search parameters used were 10 ppm peptide mass tolerance, 20 mmu fragment mass tolerance, one missed cleavage allowed, carbamidomethylation set as fixed modification and methionine oxidation and deamidation of asparagine and glutamine as variable modifications. Mascot integrated decoy database search was set to a false discovery rate (FDR) of 1% when searching was performed on the concatenated mgf files with a percolator ion score cutoff of 13 and an appropriate significance threshold p. Peptide assignment was reimported to Progenesis software. All unique peptides allocated to a protein were considered for quantification. Proteins with a ratio of at least five-fold in normalized abundance between control and ID samples were defined as differentially expressed.

Kleinwort K.J., Hauck S.M., Degroote R.L., Scholz A.M., Hölzel C., Maertlbauer E.P, & Deeg C. (2019). Peripheral blood bovine lymphocytes and MAP show distinctly different proteome changes and immune pathways in host-pathogen interaction. PeerJ, 7, e8130.

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Mass Spectrometry Proteomic Workflow

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Acquired MS spectra were imported into Progenesis software (version 2.5 Nonlinear Dynamics, Waters and analyzed as previously described^{45 (link),46 (link)}. After alignment, peak picking, exclusion of features with charge of 1 and >7 and normalization, spectra were exported as Mascot Generic files and searched against the Ensembl Horse database (Version 78) with Mascot (Matrix Science, Version 2.4.1). Peptide assignment was reimported to Progenesis Software. All unique peptides allocated to a protein were considered for quantification. Only proteins quantified with at least two peptides were included for further analysis. Abundances in individual samples were calculated by adding the single abundances of the respective peptides. Protein ratios between septin 7- or DOCK8-IP samples and the respective isotype control samples were determined by dividing these abundances. Data of DOCK8 interaction proteomics was analyzed with gene ranker (Genomatix version 3.7) for enrichment of canonical signal transduction pathways, using orthologous genes.

Schauer M., Kleinwort K.J., Degroote R.L., Wiedemann C., Kremmer E., Hauck S.M, & Deeg C.A. (2018). Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity. Scientific Reports, 8, 12332.

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