An MS/MS ion search was performed using MASCOT software (version 2.4.1, Matrix Science, Boston, MA, USA), and peptide fragment data were acquired from the peptide peaks in ESI-MS through ESI-MS/MS. Trypsin was selected as the enzyme of choice, allowing for a maximum of two potential missed cleavage sites. The instrument type was ESI-TRAP, and the peptide fragments were searched against the database using MASCOT software (version 2.4.1, Matrix Science) and FASTA search engine. The search was limited to Mus musculus taxonomy within the NCBInr and UniprotKB databases. Mass tolerance was set to ± 10 ppm for peptides and ± 0.8 Da for fragments, and high-scoring peptides were defined as those with a score greater than the default significance threshold in MASCOT (p < 0.05, peptide score > 55).
Mascot software
Manufactured by Matrix Science
Sourced in United Kingdom, United States, Japan
Mascot software is a bioinformatics tool designed for the identification of proteins from mass spectrometry data. It provides a platform for the analysis and interpretation of peptide mass fingerprinting and tandem mass spectrometry data.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Promega (11 mentions)
Proteome discoverer, by Thermo Fisher Scientific (10 mentions)
Decyder ms differential analysis software, by GE Healthcare (8 mentions)
Scaffold software, by Proteome Software (6 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (6 mentions)
C18 ziptip, by Merck Group (5 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (4 mentions)
Sequencing grade modified trypsin, by Promega (4 mentions)
Ziptip, by Merck Group (4 mentions)
Q exactive, by Thermo Fisher Scientific (4 mentions)
Nanoacquity uplc, by Waters Corporation (3 mentions)
Sequencing grade trypsin, by Promega (3 mentions)
Ltq orbitrap, by Thermo Fisher Scientific (3 mentions)
Gps explorer software, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (3 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (3 mentions)
Flexanalysis 3, by Bruker (3 mentions)
Trypsin digestion, by Promega (3 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000 nano lc system, by Thermo Fisher Scientific (3 mentions)
278 protocols using mascot software
1
Peptide Identification by MS/MS and MASCOT
2
Proteomic Analysis of Nanoparticle Toxicity
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Image analysis was carried out using an Image MasterTM 2D Platinum software (GE Healthcare Life Science, Pittsburgh, PA, USA). To compare the densities of protein spots induced by bulk or nano ZnOs, more than 25 spots were landmarked and normalized. In-gel digestion of protein spots from Coomassie Blue stained gels was performed as previously described [51 (link)]. Prior to mass spectrometric analysis, the peptide solutions were desalted using a reversed-phase column [52 (link)]. The eluted peptides were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) on a nano ACQUITY UPLC (Waters, Milford, MA, USA) directly coupled to a Finnigan LCQ DECA iontrap mass spectrometer (Thermo Scientific, Waltham, MA, USA). Spectra were acquired and processed using the MASCOT software (Matrix Science, London, UK). The individual spectra from MS/MS were processed using a SEQUEST software (Thermo Quest, San Jose, CA, USA). Only significant hits as defined by the MASCOT software (Matrix Science, London, UK) probability analysis were taken.
3
Mass Spectrometry Protein Identification
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An MS/MS ion search was assigned as the ion search preference in the MASCOT software (version 2.4.1, Matrix Science, Boston, MA, USA). Peptide fragment files were obtained from the peptide peaks in ESI–MS by ESI–MS/MS. Trypsin was selected as an enzyme with a maximum of two missed cleavage sites. ESI-TRAP was selected as the instrument type. The peptide fragments were searched based on the database using the MASCOT software (version 2.4.1, Matrix Science) and FASTA search engine, and the search was limited to Mus musculus taxonomy in NCBInr and UniprotKB/swissprot databases. The mass tolerance was set at ± 10 ppm and ± 0.8 Da for the peptides and fragments, respectively. High scoring was defined as those greater than the default significance threshold in MASCOT (p < 0.05, peptide score, > 55).
4
Proteomic Identification and Isotopic Enrichment
5
Proteomic Analysis of Yersinia pestis
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The Δail mutant was grown overnight at 28°C in LB broth with aeration. Cells were diluted 1:100 into fresh LB broth, incubated for 24 h at 37°C, and prepared as described for the supernatant protein precipitation as described above. Proteins were resolved by SDS-PAGE (72 (link)) and stained with Coomassie blue. A 2-mm-wide vertical strip spanning each lane of gel-resolved proteins was excised, divided into five parts, destained, and trypsinized (24 (link), 75 (link), 76 (link)). MS/MS analysis using a Waters Nanoacquity ultraperformance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) was performed as described previously (24 (link), 77 (link)). A ProteinLynx Global Server 2.2 and Protein Expression Informatics System software version 1.0 were used for MS/MS spectral analysis, peptide sequencing, and protein identification. MS/MS data were compared to the protein sequence databases of Y. pestis KIM from the University of Wisconsin (http://www.genome.wisc.edu/sequencing/pestis.htm ) and Y. pestis CO92 from the Sanger Institute (http://www.sanger.ac.uk/Projects/Y_pestis/ ). Results were analyzed using Mascot software (Matrix Science, London, UK). Gene identities (ID) of protein products detected were recorded compared to the Y. pestis KIM genome.
6
Identification of Myc-tagged GKRP Interactors
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Myc-tagged GKRP was immunoprecipitated from HeLa cells using an anti-Myc antibody. After immunoblot, protein bands were excised from stained one-dimensional electrophoresis gels and destained with 25 mM ammonium bicarbonate and 50% acetonitrile. In-gel digestion of dried gel pieces was performed using sequencing grade trypsin (Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate buffer overnight at 37 °C. The tryptic peptides were desalted using a GELoader tip (Eppendorf, Hamburg, Germany) packed with 1.5 μg of POROS® 20 R2 resin (PerSpective Biosystems, Ramsey, MN, USA) and applied to a C18 RP-HPLC column (75 m × 150 mm). An Agilent 1100 Series LC system was then used to separate the trypsin-digested peptides, which were eluted with a 0–40% acetonitrile gradient for 60 min. followed by analysis using a Finnigan LCQ Deca (ThermoQuest, San Jose, CA, USA) equipped with a nanoelectrospray ion source. Spray and tube lens voltages were 1.9 kV and 40 V, respectively. The capillary temperature was maintained at 250 °C at 5 V. The individual LC-MS/MS spectra were processed using TurboSEQUEST software (ThermoQuest) and the sequences were searched in NCBI databases using MASCOT software (Matrix Science Ltd., London, UK).
7
Proteomic Analysis of Tardigrade Stress Response
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Heat soluble proteomics was conducted as previously described [30 (link)]. Briefly, approximately 3000 individuals were collected from the wild and cleaned as described above and homogenized using BioMasher II (Nippi) in PBS (Nippon Gene) on ice with protease inhibitors (Roche). The lysate was heated at 92 °C for 20 min, and the soluble fraction was collected by taking the supernatant after centrifugation at 12,000 rpm for 20 min. Proteins were digested with trypsin, and tryptic peptides were separated and identified with an UltiMate 3000 nanoLC pump (Dionex Co., Sunnyvale, CA, USA) and an LTQ Orbitrap XL ETD (Thermo Electron, Waltham, MA, USA). Corresponding peptide sequences were retrieved from six frame translation data of our initial genome assembly using MASCOT software (Matrix Science) [79 (link)]. Candidates were further screened with the following conditions: (1) lack of conservation in other tardigrades or metazoans and (2) high mRNA expression (TPM > 100) in the tun state. We then predicted the structural features using the Fold Index [80 (link)] and DISOPRED [81 (link)].
8
Proteomic Analysis of Rice Proteins
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All MS/MS spectra were searched in the NCBInr Oryza sativa sequence databases and UniProtKB/Swiss‐Prot database using the MASCOT software (Matrix Science, UK; version 2.3.02). The search parameters were as follows: (1) allowed one missed cleavage in the protein trypsin digests; (2) fixed modifications of carbamidomethylation at cysteine, variable modifications of oxidation at methionine and iTRAQ 8‐plex at tyrosine; (3) peptide tolerance was set at ± 0.02 Da and MS/MS tolerance was set at ± 0.05 Da. The peptide charge was set as Mr, and the monoisotopic mass was chosen. The iTRAQ 8‐plex was chosen for quantification during the search. The search results were filtered before data exportation. The filters were used for protein identification with the following options: significance threshold of p < 0.05 (with 95% confidence) and ion score or expected cutoff of < 0.05 (with 95% confidence). For protein quantification, the filters were set as follows: (1) “median” was chosen for the protein ratio type; (2) the minimum precursor charge was set to 2, minimum peptides were set to 2, and only unique peptides were used for quantitation; and (3) normalization by median intensities, and outliers were removed automatically.
9
Protein Identification and Quantification
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The MS/MS spectra were searched against the SwissProt human database (www.uniprot.org , 20,227 entries) using Mascot software (version 2.3.02; Matrix Science, Ltd., London, UK). The parent and fragment ion mass tolerances were 0.050 Da. The carbamidomethylation of cysteine was set as a fixed modification, and the number of miscleavage sites allowed was ≤2. Scaffold software (version Scaffold_4.0.7, Proteome Software, Inc., Portland, OR, USA) was used to filter the results. Protein identification was accepted at a false discovery rate (FDR) <1.0% by analyzing the levels of proteins and peptides formed by ≥2 unique peptides. The identified proteins were quantified based on iTRAQ reporter ion intensities of unique peptides. The proteins containing quantification values in all channels in all three runs were considered suitable for quantification.
10
Protein Sequence Identification
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These data were then used to search the NCBI non-identical protein sequence database using MASCOT software (Matrix Science), and statistically significant hits were recorded together with the number of peptides and percentage coverage of the protein.
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