After 2D electrophoresis, spots positive for AX or AX-B were located in the Sypro-Ruby stained gels, processed by trypsin digestion and analyzed by MS (7) . MS data were used to interrogate the NCBI non-redundant protein database SwissProt 2014_03 (542782 sequences; 193019802 residues) using MASCOT software 2.3 (Matrix Science).
Mascot software 2
Manufactured by Matrix Science
Sourced in United Kingdom
MASCOT Software 2.2 is a bioinformatics tool designed for the identification and characterization of proteins from mass spectrometry data. It provides a comprehensive platform for the analysis of peptide and protein sequences.
Automatically generated - may contain errors
Lab products found in correlation
Ultraflex maldi tof tof mass spectrometer, by Bruker (2 mentions)
Flexanalysis software, by Bruker (1 mentions)
Autoflex 3 tof tof mass spectrometer, by Bruker (1 mentions)
Biotools 3, by Bruker (1 mentions)
Q exactive instrument, by Thermo Fisher Scientific (1 mentions)
Proteome discover, by Thermo Fisher Scientific (1 mentions)
Proteome discover 1, by Thermo Fisher Scientific (1 mentions)
9 protocols using mascot software 2
1
Proteomic Identification of Proteins
2
MALDI-TOF Mass Spectrometry Protein Identification
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Samples were analysed with an Autoflex III TOF/TOF mass spectrometer (Bruker-Daltonics, USA). Typically, 1000 scans for peptide mass fingerprinting (PMF) and 2000 scans for MS/MS were collected. Automated analysis of mass data was performed using FlexAnalysis software (Bruker-Daltonics, USA). Internal calibration of MALDI-TOF mass spectra was performed using two trypsin autolysis ions with m/z 842.510 and m/z 2211.105; as for MALDI-MS/MS, calibrations were performed with a fragment ion spectrum obtained from the proton adducts of a peptide mixture covering the m/z 700-4000 region. The typical error observed in mass accuracy for calibration was usually below 50 ppm. MALDI-MS and MS/MS data were combined through the BioTools 3.0 program (Bruker-Daltonics, USA) to interrogate the NCBI non-redundant protein database SwissProt 2014_03 (542782 sequences; 193019802 residues) using MASCOT software 2.3 (Matrix Science, UK). Relevant search parameters were set as follows: enzyme, trypsin; fixed modifications, carbamidomethyl (C); oxidation (M); 1 missed cleavage allowed; peptide tolerance, 50 ppm; MS/MS tolerance, 0.5 Da.
Peptide mass fingerprinting and fragmentation by MS-MALDI-TOF was carried out in the Proteomics and Genomics Facility (CIB-CSIC), a member of ProteoRed-ISCIII network.
Peptide mass fingerprinting and fragmentation by MS-MALDI-TOF was carried out in the Proteomics and Genomics Facility (CIB-CSIC), a member of ProteoRed-ISCIII network.
3
Automated Protein Identification in Rat Tissues
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Manually excised gel plugs were subjected to an automated platform for the identification of gel-separated proteins69 as described. An Ultraflex MALDI-TOF-TOF mass spectrometer (Bruker Daltonik) was used to acquire both PMF and fragment ion spectra, resulting in confident protein identifications based on peptide mass and sequence information. Database searches in the Swiss-Prot and NCBI primary sequence database restricted to the taxonomy rattus norvegicus were performed using the MASCOT Software 2.2 (Matrix Science). Carboxamidomethylation of Cys residues was specified as fixed and oxidation of Met as variable modifications. One trypsin missed cleavage was allowed. Mass tolerances were set to 100 ppm for PMF searches and to 100 ppm (precursor ions) and 0.7 Da (fragment ions) for MS/MS ion searches. The minimal requirement for accepting a protein as identified was at least one peptide sequence match above identity threshold in addition to at least 20% sequence coverage in the PMF.
4
Automated Protein Identification Protocol
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Manually excised gel plugs were subjected to an automated platform for the identification of gel-separated proteins as described earlier [20 (link),21 (link)]. The mass spectrometric analysis was performed according to our standard protocol. Database searches in the Swiss-Prot and NCBI primary sequence database were performed using the MASCOT Software 2.2 (Matrix Science, London, United Kingdom). The minimum requirement for accepting a protein as identified was at least one peptide sequence match above the identity threshold in addition to at least 20% sequence coverage in the peptide mass fingerprint (PMF).
5
Automated Protein Identification from Gel Plugs
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Manually excised gel plugs were subjected to an automated platform for the identification of gel-separated proteins as described recently [20 (link)]. An Ultraflex MALDI-TOF-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) was used to acquire both peptide mass fingerprinting and fragment ion spectra, resulting in confident protein identifications based on peptide mass and sequence information. Database searches in the Swiss-Prot primary sequence database restricted to the taxonomy Homo sapiens were performed using the MASCOT Software 2.2 (Matrix Science, London, UK). Carboxamidomethylation of Cys residues was specified as fixed modification and oxidation of Met residues as variable modifications. One trypsin-missed cleavage was allowed. Mass tolerances were set to 100 ppm for peptide mass fingerprinting searches and to 100 ppm (precursor ions) and 0.7 Da (fragment ions) for MS/MS ion searches. The minimal requirement for accepting a protein as identified was at least one peptide sequence match above identity threshold in addition to at least 20% sequence coverage in the peptide mass fingerprinting.
6
Proteomics Analysis of Haemonchus contortus
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Samples collected by Co-IP were digested with trypsin and analysed by shotgun LC–MS/MS using a Q Exactive instrument (Thermo Finnigan, San Jose, CA, USA) at Shanghai Bio Profile Technology Company Ltd. (Shanghai, China) as previously described [32 (link)]. For proteomic identification, based on the corresponding UniProt database (UniProt_Haemonchus_contortus_4037_20191119.fasta), Mascot software 2.2 was used for peptide mass fingerprinting and peptide sequence tagging (v.2.2, Matrix Science, London, UK). Carbonamidomethyl (C) was identified as a fixed modification, and oxidation (M) was identified as a variable modification, allowing less than two missed cleavages. Furthermore, all the identified peptides were screened with a false discovery rate (FDR) ≤ 0.01 and filtered for a score ≥ 20. The peptide mass tolerance was 20 ppm, and the fragment mass tolerance was 0.1 Da. Protein identification in the MASCOT search was applied to UniProtKB and QuickGO. Gene Ontology (GO) classification (molecular function, biological process and cellular component terms) was carried out using Blast2GO based on the BLASTP results.
7
Quantitative Proteomic Analysis of Sus scrofa
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The raw mass data were converted using Proteome Discover 1.3, and protein identification was performed using Mascot Software 2.3.02 (Matrix Science, London, UK) against the Uniprot Sus scrofa database (33,785 sequences). For the protein identification, the following options were used: enzyme = trypsin; missed cleavage = 1; peptide mass tolerance = ±0.05 Da; fragmented ions tolerance = ±0.1 Da; fixed modification = Carbamidomethyl (C), iTRAQ 8 plex (N-term) and iTRAQ 8 plex (K); potential variable modification = Gln→pyro-Glu (N-term Q), Oxidation (M) and Deamidation (NQ). The protein with at least one unique peptide, which had a significant score at the 95% confidence interval through the Mascot probability analysis, was counted as identified. The quantitative protein ratios were weighted and normalised by the median ratio in Mascot. The protein with three abundance ratios >1.2 (or < 0.83) and p-value < 0.05 was considered as DAP.
8
Mass Spectrometry Analysis of Fractionated Peptides
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The fractionated peptides were analyzed using a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific). Raw data were processed with Proteome Discover version 1.4 (Thermo Fisher Scientific) and searched with Mascot software 2.3.02 (Matrix Science, London, United Kingdom) against a personalized transcriptome database for P. antidotale, with a precursor mass tolerance of 15 ppm, a fragment ion mass tolerance of 20 mm, and strict trypsin specificity. This allowed up to two missed cleavages, carboxyamidomethylation modification on cysteine residues as fixed modification, and oxidation of methionine residues and phosphorylation of serine, threonine, and tyrosine residues as variable modifications. Proteins with at least one unique peptide and a threshold of FDR having a p-value of < 0.01 were considered positive identification. A fold-change of ≥ 1.2 (p < 0.05) was taken as a threshold to identify differentially expressed proteins (DEPs).
9
Cotton Proteome Identification and Quantification
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Raw mass data were processed with Proteome Discover 1.3 (Thermo Fisher Scientific) and searched with Mascot software 2.3.02 (Matrix Science, London, U.K.) against the database downloaded from ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/plant/Gossypium_raimondii/latest_assembly_versions/ . The search parameters were set as follows: trypsin was specified as the digestion enzyme, carbamidomethylation of cysteine was set as a fixed modification, oxidation of methionine was set as a variable modification, peptide tolerance was set to 10 ppm, and MS/MS tolerance was set to 0.05 Da. Proteins with at least 2 unique peptides and a threshold p-value (with 95% confidence) of < 0.05 were qualified for further quantitative data analysis. For protein abundance ratios, fold-changes of ≥1.2 (or ≤0.83) and p-values of < 0.05 were taken as thresholds to identify significant changes.
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