Mascot search engine

Manufactured by Matrix Science

Sourced in United Kingdom, United States, Germany

The Mascot search engine is a software tool designed for the identification of proteins from mass spectrometry data. It provides a comprehensive solution for the analysis and interpretation of proteomic data.

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Lab products found in correlation

457 protocols using mascot search engine

Protein Identification via Mascot Search

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Raw data files were pre-processed with Mascot Distiller software (version 2.6, MatrixScience, London, UK). The obtained peptide masses and fragmentation spectra were matched to the NCBInr database (147075655 sequences; 53900923684 residues), with a Nematoda filter (928973 sequences) using the Mascot search engine (MatrixScience, London, UK, Mascot Server 2.5). The following search parameters were applied: enzyme specificity was set to trypsin, peptide mass tolerance to ± 30 ppm and fragment mass tolerance to ± 0.1 Da. The protein mass was left as unrestricted, and mass values as monoisotopic with one missed cleavage being allowed. Alkylation of cysteine by carbamidomethylation as fixed and oxidation of methionine was set as a variable modification. Protein identification was performed using the Mascot search engine (MatrixScience), with the probability-based algorithm. An expected value threshold of 0.05 was used for analysis, which means that all peptide identifications had less than 1 in 20 chance of being a random match.
All proteins identified in the Mascot search were assigned to the UniProtKB database (https://www.uniprot.org/) and QuickGO (http://www.ebi.ac.uk/QuickGO/) and classified in gene ontology (GO) in accordance with its molecular function, biological process and cellular component information.

Grzelak S., Stachyra A., Stefaniak J., Mrówka K., Moskwa B, & Bień-Kalinowska J. (2020). Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella. PLoS ONE, 15(11), e0241918.

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Neuron-Specific Acetylome Profiling

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The neuron-specific acetylome was determined as previously described (Pehar et al., 2012b (link)). In brief, adult neurons were isolated with the gentle MACS Dissociator (Miltenyi Biotec). Proteins were digested with trypsin before high-resolution high-accuracy LC-MS/MS analysis at the Mass Spectrometry facility at the University of Wisconsin-Madison. Peptides and proteins were identified with the Mascot search engine (Matrix Science) via automated database searching of all tandem mass spectra. The output of the MS analysis was further processed to select proteins that insert into the secretory pathway.

Hullinger R., Li M., Wang J., Peng Y., Dowell J.A., Bomba-Warczak E., Mitchell H.A., Burger C., Chapman E.R., Denu J.M., Li L, & Puglielli L. (2016). Increased expression of AT-1/SLC33A1 causes an autistic-like phenotype in mice by affecting dendritic branching and spine formation. The Journal of Experimental Medicine, 213(7), 1267-1284.

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Differential Protein Expression Analysis

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The raw MS/MS data were converted to MGF format and files were searched using the Mascot search engine (Matrix Science, London, UK; version 2.3.02) to identify and quantify proteins. IQuant software was utilized for iTRAQ data quantification. Proteins with an average ratio of more than 1.2 fold and a p-value less than 0.05 were determined as differentially expressed proteins (DEPs). All proteins with FDR less than 1% were subjected to analyses including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Based on the DEPs, we further analyzed the results of the GO enrichment and KEGG pathway enrichment analyses. In addition, we also analyzed interactions between the significant KEGG pathways and performed protein-protein interaction (PPI) analysis with STRING (Version 11.0) and Cytoscape software (Version 3.8.2).

Li B., Xian X., Lin X., Huang L., Liang A., Jiang H, & Gong Q. (2022). Hypoxia Alters the Proteome Profile and Enhances the Angiogenic Potential of Dental Pulp Stem Cell-Derived Exosomes. Biomolecules, 12(4), 575.

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Proteomic Analysis of Differentially Expressed Proteins

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The significantly differentially expressed protein spots were excised from the gels and destained with 50% acetonitrile (ACN) in 100 μL of 25 mM ammonium bicarbonate. After destaining, tryptic digestion was performed according to a previously described method [49 (link)]. The resulting peptide mixtures were analyzed with an UltiMate 3000 RSLCnano System (Ultimate 3000, Dionex, USA) coupled to a micrOTOF-Q II ESI-Qq-TOF mass spectrometer (Bruker Daltonics, Germany). The MS/MS spectra data from each sample were searched against the NCBI databases using the MASCOT search engine (Matrix Science, London, UK) (http://www.matrixscience.com/) [50 (link)].

Pichitpunpong C., Thongkorn S., Kanlayaprasit S., Yuwattana W., Plaingam W., Sangsuthum S., Aizat W.M., Baharum S.N., Tencomnao T., Hu V.W, & Sarachana T. (2019). Phenotypic subgrouping and multi-omics analyses reveal reduced diazepam-binding inhibitor (DBI) protein levels in autism spectrum disorder with severe language impairment. PLoS ONE, 14(3), e0214198.

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Proteomic Analysis via MALDI-TOF MS

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Spots of interest were excised and digested in-gel with trypsin for further protein identification according to previously described procedures [51 (link)]. After digestion, the tryptic peptides were acidified with 0.5% TFA and loaded onto an MTP AnchorChip^TM 600/384 TF (Bruker-Daltonik, Bremen, Germany). MS analysis was performed on an Ultraflex^TM MALDI-TOF mass spectrometer (Bruker-Daltonik). Monoisotopic peptide masses were assigned and used for database searches with the MASCOT search engine (Matrix Science, London, UK). Search parameters were set as follows: a maximum allowed peptide mass error of 50 ppm, and consideration of one incomplete cleavage per peptide.

Wu T.H., Lin T.Y., Yang P.M., Li W.T., Yeh C.T, & Pan T.L. (2024). Scutellaria baicalensis Induces Cell Apoptosis and Elicits Mesenchymal–Epithelial Transition to Alleviate Metastatic Hepatocellular Carcinoma via Modulating HSP90β. International Journal of Molecular Sciences, 25(5), 3073.

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Protein Identification by LC-MS/MS

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After stained by Coomassie blue, the SDS‐PAGE gels were divided and cut into seven parts that were sent to Huada Protein Research Center (HPRC) for LC‐MS/MS analysis. MS/MS data were searched by HPRC against ncbi_insecta_201401 201401 (1,786,564 sequences; 642,693,167 residues) database using the Mascot search engine (Matrix Science, London, UK). Search parameters are provided by HPRC as follows: type of search – MS/MS ion search; enzyme – trypsin; fixed modifications – carboxymethyl (C); variable modifications – Gln>pyro‐Glu (N‐term Q) and oxidation (M); mass values – monoisotopic; protein mass – unrestricted; peptide mass tolerance – ±15 ppm; fragment mass tolerance – ±20 mmu; max missed cleavages – 1; instrument type – default; number of queries – 9983. Peptide identifications were accepted if they could be established at greater than 50% probability as specified by the PeptideProphet algorithm (Keller et al. 2002). Protein identifications were accepted if they could be established at greater than 90% probability and contained at least two identified peptides. Protein probabilities were assigned by the ProteinProphet algorithm (Nesvizhskii et al. 2003).

Zhou Z., Wang Z., Liu Y., Liang G., Shu C., Song F., Zhou X., Bravo A., Soberón M, & Zhang J. (2016). Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles from Helicoverpa armigera by an improved pull‐down assay. MicrobiologyOpen, 5(4), 659-669.

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iTRAQ Proteomics Analysis of Rat Samples

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The raw data were processed and converted using the Mascot search engine (Matrix Science, v.2.3.02) and maascot generic file with Proteome Discoverer (Thermo Scientific, v1.4.1.14). A total of 29 982 sequences of tandem-mass spectra were retrieved using a rat reference proteome. The mass error of precursor ions was set at 10 PPM and fragment ions at 0.02 Da. Enzyme specificity was determined by trypsin and 2 missed cleavages were allowed. The iTRAQ 8-plex tag on Lys, the N-terminal peptide, and the carbamidomethylation tag on Cys were specified as fixed modifications, whereas iTRAQ 8-plex tag on Tyr and oxidation on Met were specified as variable modification. False discovery rates (FDR) were estimated from a Decoy (reverse) database. The algorithm percolator and PSMs (Peptide-Spectrum Match) were used to revalue the calculated results using a P value <0.05 and an e-value <0.05. Thus, to be included, each protein must contain at least 2 identical unique peptides. The type of protein ratio was weighted, and all were normalized to the median.

Liu S., Kang Y., Zhang C., Lou Y., Li X., Lu L., Qi Z., Jian H, & Zhou H. (2020). Isobaric Tagging for Relative and Absolute Protein Quantification (iTRAQ)-Based Quantitative Proteomics Analysis of Differentially Expressed Proteins 1 Week After Spinal Cord Injury in a Rat Model. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e924266-1-e924266-12.

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Proteomic analysis of purified proteins

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Purified proteins were prepared for mass spectrometric analysis by in solution enzymatic digestion, without prior reduction and alkylation. Protein samples were digested with trypsin or elastase (Promega), both at an enzyme to protein ratio of 1:20. The resulting peptides were analysed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC (ThermoScientific Dionex) to deliver a flow of approximately 300 nl/min. A C18 Acclaim PepMap100 5 µm, 100 µm x 20 mm nanoViper (ThermoScientific Dionex), trapped the peptides prior to separation on a C18 Acclaim PepMap100 3 µm, 75 µm x 250 mm nanoViper (ThermoScientific Dionex, San Jose, USA). Peptides were eluted with a 90 min gradient of acetonitrile (2% to 50%). The analytical column outlet was directly interfaced via a nano-flow electrospray ionization source, with a hybrid quadrupole orbitrap mass spectrometer (Q-Exactive Plus Orbitrap, ThermoScientific). LC-MS/MS data were then searched against an in house LMB database using the Mascot search engine (Matrix Science) 72 (link), and the peptide identifications validated using the Scaffold program (Proteome Software Inc.) 73 (link). All data were additionally interrogated manually.

Alfieri C., Chang L., Zhang Z., Yang J., Maslen S., Skehel M, & Barford D. (2016). Molecular basis of APC/C regulation by the spindle assembly checkpoint. Nature, 536(7617), 431-436.

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Proteomic analysis of purified proteins

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Alfieri C., Chang L., Zhang Z., Yang J., Maslen S., Skehel M, & Barford D. (2016). Molecular basis of APC/C regulation by the spindle assembly checkpoint. Nature, 536(7617), 431-436.

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Proteomic Analysis of AlgoCIS Samples

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The purified AlgoCIS samples were sent in solutions to the Functional Genomics Center Zürich (FGCZ), which performed the mass spectrum and the subsequent data analysis. The samples were first digested by trypsin. These digested samples were dried and dissolved in 20 μl ddH₂O with 0.1% formic acid. The samples were transferred to autosampler vials for liquid chromatography–mass spectrometry analysis (LC–MS/MS). The samples were diluted at the ratio 1:40, with 1 μl of each sample being injected on a nanoAcquity UPLC coupled to a Q-Exactive mass spectrometer (ThermoFisher).
The acquired MS data were converted to a Mascot Generic File format and were processed for identification using the Mascot search engine (Matrixscience). In addition, the acquired MS data were imported into PEAKS Studio (Bioinformatic Solutions) and were searched against the Algoriphagus machipongonensis database. The results were visualized by Scaffold software.

Xu J., Ericson C.F., Lien Y.W., Rutaganira F.U., Eisenstein F., Feldmüller M., King N, & Pilhofer M. (2022). Identification and structure of an extracellular contractile injection system from the marine bacterium Algoriphagus machipongonensis. Nature Microbiology, 7(3), 397-410.

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