Trypsin samples were digested and the substrate CCA was mixed and applied to the target sample for natural drying. The protein chip was placed into MALDI-TOF/TOF MS to detect the peptide fragment of enzymolysis, and Mascot search software (Matrix Science, Ltd., London, UK) was used to retrieve the proteins that may match with the identified peptides and amino acids in the SwissProt database (6 (link)).
Mascot search software
Manufactured by Matrix Science
Sourced in United Kingdom
The Mascot search software is a powerful tool used for the identification and characterization of proteins from mass spectrometry data. It provides fast and accurate database searching capabilities, allowing users to analyze and interpret complex proteomic datasets.
Automatically generated - may contain errors
Lab products found in correlation
Proteome discoverer 1, by Thermo Fisher Scientific (4 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Pierce crosslink ip kit, by Thermo Fisher Scientific (1 mentions)
Pierce classic ip kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using mascot search software
1
MALDI-TOF/TOF MS Peptide Identification
2
Quantitative Proteomics Data Analysis
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The raw dates from Q Exactive were submitted to Mascot search software (version 2.2.1; Matrix Science, London, UK) and Proteome Discoverer 1.4 (Thermo Electron, San Jose, CA, USA) for date filtering and proteins identification. Furthered analysis was performed according to false discovery rate (FDR) ≤0.01 using Proteome Discoverer 1.4. Protein identification was supported by at least one unique peptide identified using the Peptide Prophet algorithm. Proteins with fold change >1.2 were retained. Hierarchical clustering analysis was performed by MeV 4.7 software, and Pearson’s correlation analysis was used for the distance matrix and the Ward’s linkage.
3
Peptide Identification and Quantification
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The peptides were subjected to nano-electrospray ionization followed by tandem mass spectrometry (MS/MS) in a Q-TOF coupled online to the HPLC system. The 3 most intense precursors from MS1 spectra were applied for MS2. Survey scans were acquired from m/z 50 to 3000. The original MS/MS file data were collected with micro TOF-Q control (Bruker, Germany) and submitted to DataAnalysis4.0, from which raw data was converted to MGF file. The Mascot search software (Matrix Science, London, UK) were used to identify and quantify the peptides. The Mascot search parameters were as follows: Enzyme: trypsin; Database: NCBInr_human; Peptide Charge:(1+, 2+ and 3+); Instrument: ESI-QUAD-TOF; Mass values: monoisotopic; Peptide mass tolerance: ±1Da; Fragment mass tolerance: ±1Da; Sample Type: iTRAQ 8plex (Peptide Labeled); Data format: Mascot generic; Fixed modification: carboxymethyl (C); Variable modifications: Gln->pyro-Glu(N-term Q), Oxidation(M), iTRAQ8plex(K), iTRAQ8plex(Y), iTRAQ8plex (N-term).
To reduce the probability of false peptide identification, only peptides with significance scores (≥ 40) at the 95% confidence interval were counted as identified. At least one unique peptide was involved in each confidently identified protein. The fold change cutoff ratio < 0.8 or >1.2 was determined to designate differential expression proteins (p < 0.05) [37 (link)].
To reduce the probability of false peptide identification, only peptides with significance scores (≥ 40) at the 95% confidence interval were counted as identified. At least one unique peptide was involved in each confidently identified protein. The fold change cutoff ratio < 0.8 or >1.2 was determined to designate differential expression proteins (p < 0.05) [37 (link)].
4
Neuropeptide Identification in P. melas
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The MS/MS data were processed to produce pick lists for searching in the SwissProt database using the MASCOT search software (© Matrix Science 2021, http://www.matrixscience.com ). The mass tolerance of the parent and fragments for MS/MS data search was set at 20 ppm and 0.20 Da, respectively. During database search the following query were considered “Metazoa (Animals)” taxonomy and choosing “noCleave”. A peaklist of 50 ions of 10% higher intensity than the noise level was generically used for database searching. Methionine oxidation was included in the variable modifications. Although several MS/MS spectra showed intense and well-resolved ion signals, all spectra were manually checked to validate MASCOT results. Neuropeptide homologs are determined by Domain Enhanced Lookup Time Accelerated Basic Local Alignment Search Tool (DELTA-BLAST), limiting the searches to homologs in Drosophila melanogaster. Network analysis was performed by STRING (Search Tool for the Retrieval of Interacting Genes) software (v. 11) (http://stringdb.org/ ). NeuroPIpred was used to evaluate potential neuropeptides in P. melas (https://webs.iiitd.edu.in/raghava/neuropipred/ ).
5
Peptide Identification in Borrelia Proteome
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Raw data files were processed using Proteome Discoverer 1.4 software (Thermo Scientific). Peak lists were searched using the MASCOT search software (Matrix Science) against the database B. burgdorferi. Database searches were performed with the following parameters: two missed trypsin cleavage sites allowed; variable modifications: carbamidomethylation on cysteine, oxidation on methionine, and lysine acetylation. The parent-ion and daughter-ion tolerances were 5 ppm and 0.35 Da, respectively. False discovery rate (FDR) threshold for identifications was specified at 1% (for proteins and peptides). For each identification, we looked carefully at the ion score (above 19), the peptide rank, the daughter ions (b- and y-fragment ion series), and the expectation value (E-value, ≤0.05). We checked manually all spectra containing acetylation to ensure both the location of the PTM and the peptide sequence (Supplementary Figure 5 ).
6
Banana Protein Identification by MS
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The MS data were searched using MASCOT search software (Version 2.6.1, Matrix Science, London, UK) and SEQUEST HT search algorithms against the Musa acuminata (Banana) UniProtKB protein database (2018-12-04) using Proteome Discoverer 2.2 (Version 2.2.0.388; Thermo Fisher Scientific, USA) using the following workflow: spectrum files RC, spectrum selector, MASCOT, SEQUEST HT search nodes, percolator, ptmRS, and minor feature detector nodes. The parameters of search were set as follows: Methionine oxidation as a variable modification and cysteine carbamidomethylation as a fixed modification, MS and MS/MS tolerances of 10 ppm and 0.6 Da, respectively, and one missed trypsin cleavage. Target-decoy database searches were used to calculate the false discovery rate (FDR). For peptide identification, FDR was set at 1%.
7
Carbamylated Peptide Identification in ACa-FCS Sera
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To perform proteomic analysis from VErA sera samples known to be ACa-FCS-positive, protein concentration was determined using the standard Bradford method (Biorad). Enzymatic digestion of proteins in sera samples has been described previously (14 (link)). Identification of carbamylated peptides was performed with a classic setup of mass spectrometry analysis using a Q-exactive Plus (ThermoScientific) equipped with a nanoESI source, as described previously (15 (link)). All spectra obtained were exported in “raw” format to identify peptides and proteins with Proteome Discoverer 1.4 software (Thermo Scientific). Peak lists were searched using MASCOT search software (Matrix Science) against the human Swissprot database with the following parameters: one miss cleavage site allowed, carbamidomethylation on cysteine, oxidation on methionine, and carbamylation on lysine as variable modifications. The parent-ion and daughter-ion tolerances were 5ppm and 0.02Da respectively.
Proteomic data deposit on ProteomeXchange via the PRIDE database (Project accession: PXD028121).
Proteomic data deposit on ProteomeXchange via the PRIDE database (Project accession: PXD028121).
8
Biotin-Conjugated SL-13R Interactome Profiling
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UCB CD34+ cells were cultured in medium with or without biotin-conjugated SL-13R. After 12 hr, cells were collected and lysed with ice cold IP Lysis/Wash Buffer from the Pierce Crosslink IP Kit (Thermo Fisher Scientific, Waltham, MA, USA). Immunoprecipitation was performed following manufacturer’s instruction of Pierce Classic IP kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Briefly, 1 mg of protein was incubated with 2 μg of murine anti-biotin IgG2a (BioLegend, San Diego, CA, USA) at 4 °C overnight. To capture protein-peptide-antibody complex, protein A/G agarose beads were added and incubated at 4 °C for one hour. The beads were then washed three times with IP lysis/wash buffer, once with conditioning buffer and subjected to elution buffer pH 2.8. After neutralizing low pH with 1 M Tris pH 9.5, proteins were digested by trypsin and resulting peptides were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) (Waters, Tokyo, Japan). After immunoprecipitation, LC-MS/MS analysis and datasets were analyzed using MASCOT Search software (Matrix Science, Tokyo, Japan).
9
Mascot-based Proteomic Data Analysis
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Raw data files were processed using Proteome Discoverer 1.4 software (Thermo Scientific). Peak lists were searched using the Mascot search software (Matrix Science, version 2.2.04) against the database Uniprot with the taxonomy Mus musculus. Database searches were performed with the following parameters: two missed trypsin cleavage sites allowed; variable modifications: carbamidomethylation on cysteine, and oxidation on methionine. The parent-ion and daughter-ion tolerances were 10 ppm and 0.5 Da, respectively. False discovery rate (FDR) threshold for identifications was specified at 1% (for proteins and peptides).
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