The samples were added to 5 µl of 0.1% TFA for re-suspension. The TFA was mixed at a 1:1 ratio with α-cyano-4-hydroxy-trans-cinnamic acid in 50% ACN. The mixed solution (1 µl) was added to a sample target plate. Using an ABI 4800 MALDI TOF/TOF Plus mass spectrometer, peptide MS and MS/MS were performed (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Using GPS Explorer™ software (version 3.6; Applied Biosystems; Thermo Fisher Scientific, Inc.), a search (MS+MS/MS) was performed. The time of flight spectra recorded the positive ion reflector mode in a mass range of 800-4,000 Da. According to the MS and MS/MS spectra, proteins were successfully determined, with a confidence interval of ≥95%, using the Mascot V2.3 search engine (Matrix Science, London, UK). The other parameters were set as follows: NCBI-Animals database; 100 ppm for precursor ion tolerance; fixed modifications of carbamidomethyl; 0.3 Da for fragment ion tolerance; and partial modifications of acetyl and oxidation.
Gps explorer software
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
GPS Explorer software is a data analysis tool that enables users to process and visualize geospatial data. It supports a variety of file formats and provides functions for mapping, spatial analysis, and data management.
Automatically generated - may contain errors
Lab products found in correlation
Mascot software, by Matrix Science (3 mentions)
4700 proteomics analyzer, by Thermo Fisher Scientific (3 mentions)
Mascot, by Matrix Science (3 mentions)
4700 maldi tof tof proteomics analyzer, by Thermo Fisher Scientific (3 mentions)
Mascot search engine, by Matrix Science (2 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
4700 proteomicsanalyzer tof tof, by Thermo Fisher Scientific (2 mentions)
Mascot program, by Matrix Science (2 mentions)
Ipgphor 2 apparatus, by Cytiva (2 mentions)
Mascot v2, by Matrix Science (1 mentions)
Abi 4800 maldi tof tof plus mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (1 mentions)
Triple tof 5600 system, by AB Sciex (1 mentions)
Tof tof ion optics, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Mascot search program, by Matrix Science (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Anti he4 antibody, by Santa Cruz Biotechnology (1 mentions)
Data explorer software, by Thermo Fisher Scientific (1 mentions)
Mascot algorithm, by Matrix Science (1 mentions)
21 protocols using gps explorer software
1
MALDI-TOF/TOF for Protein Identification
2
Quantitative Proteomic Analysis of PAB Treatment
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Each gel consisted of three samples run concurrently: one 30 min. before PAB sample (Control group), one 48 hrs after PAB sample (Test group), and an internal standards. Plasma from Control group and Test group was labelled with either Cy3 (green) or Cy5 (red). The three samples (Cy2, Cy3, Cy5) to be run on a single gel were then combined and applied to immobilized pH gradient strips. Gels were scanned and imported into DeCyder 6.5 differential analysis software. All gels were standardized with the internal standard (Cy2) images. A visible protein stain, Coomassie Brilliant Blue G250 stain was applied to allow visual inspection of the protein spots for mass spectrometic analysis.
Upon sonication, peptides were extracted and dried completely by centrifugal lyophilization. Peptide mixtures were dissolved, and 1 mL of peptide solution was mixed with 1 mL of matrix before spotting on the Matrix-assisted laser desorption/ionization sample plate. The peptide mass fingerprinting and sequence tag data were evaluated with GPS Explorer™ software (Applied Biosystems, Foster City, CA, USA). Mass spectrometry (MS)/MS spectra were submitted to the Biotechnology Information database IPI HUMAN 3.83 to generate ion scores via the Mascot search engine (Matrix Science Inc, Boston, MA, USA) 3 (link).
Upon sonication, peptides were extracted and dried completely by centrifugal lyophilization. Peptide mixtures were dissolved, and 1 mL of peptide solution was mixed with 1 mL of matrix before spotting on the Matrix-assisted laser desorption/ionization sample plate. The peptide mass fingerprinting and sequence tag data were evaluated with GPS Explorer™ software (Applied Biosystems, Foster City, CA, USA). Mass spectrometry (MS)/MS spectra were submitted to the Biotechnology Information database IPI HUMAN 3.83 to generate ion scores via the Mascot search engine (Matrix Science Inc, Boston, MA, USA) 3 (link).
3
Proteomic Identification of Differentially Expressed Proteins
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The gels of 2DE were recorded as digitalized images using a Calibrated Densitometer (GS-800, Bio-Rad, USA), and the search for differentially expressed proteins was carried out using the software of PDQuest Advanced 8.0.1. Protein spots were obtained from the silver-stained gels, detained, and subjected to tryptic digestion as described by Gokulakannan and Niehaus
[23 (link)] for MS. The peptides mixtures were re-dissolved in matrix solution, dried, and analyzed by a 4800 matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF)(Applied Biosystems, Carlsbad, CA, USA). Combined MS and MS/MS spectra were subjected to MASCOT (version 2.1, Matrix Science, London, UK) by the GPS Explorer software (version 3.6, Applied Biosystems) and searched with the following parameters, National Center for Biotechnology Information Non-redundant (NCBInr) and EST databases. Known contaminant ions (tryptic auto-digest peptides) were excluded. The individual MS/MS spectrum, with a statistically significant (confidence interval ≥95%) ion scores (based on MS/MS spectra), was accepted.
[23 (link)] for MS. The peptides mixtures were re-dissolved in matrix solution, dried, and analyzed by a 4800 matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF)(Applied Biosystems, Carlsbad, CA, USA). Combined MS and MS/MS spectra were subjected to MASCOT (version 2.1, Matrix Science, London, UK) by the GPS Explorer software (version 3.6, Applied Biosystems) and searched with the following parameters, National Center for Biotechnology Information Non-redundant (NCBInr) and EST databases. Known contaminant ions (tryptic auto-digest peptides) were excluded. The individual MS/MS spectrum, with a statistically significant (confidence interval ≥95%) ion scores (based on MS/MS spectra), was accepted.
4
Quantitative Proteomic Analysis of Viral Infection
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The LC-MS analysis was performed on a Triple-TOF 5600 system (AB SCIEX). Mass spectra were collected (m/z 400–1250) in high resolution (> 30,000), using 250 ms per spectrum. A maximum of 50 precursors in a cycle were selected for fragmentation from each mass spectrum. Tandem mass spectra were harvested in high sensitivity mode (resolution > 15,000).
The analyses of peptide and protein identifications and iTRAQ quantification were performed on GPS Explorer software (Applied Biosystems) using MASCOT search engine (Matrix Science). The NCBI database (http://www.ncbi.nlm.nih.gov/protein/?term=txid9615[Organism:noexp] ) was selected for analysis. Cysteine methane thiolation, iTRAQ-labeled lysine, and N-terminal iTRAQ labeling were selected as fixed modifications in the data analysis (Zieske, 2006 (link)).
Two-sample unequal variant-test was selected to analyze the difference between mock-infected and infected samples. The different proteins were corresponded to a P-value < 0.05. Threshold for iTRAQ was defined according to 95% CV+1.
The analyses of peptide and protein identifications and iTRAQ quantification were performed on GPS Explorer software (Applied Biosystems) using MASCOT search engine (Matrix Science). The NCBI database (
Two-sample unequal variant-test was selected to analyze the difference between mock-infected and infected samples. The different proteins were corresponded to a P-value < 0.05. Threshold for iTRAQ was defined according to 95% CV+1.
5
Protein Identification in Green Plants
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The primary and secondary mass spectrum data were analyzed with the GPS Explorer software (Version 3.6, Applied Biosystems, Lincoln Centre Drive Foster City, CA, USA). Database searches were performed with the MASCOT software (Version 2.1.03, Matrix Science, London, UK) using the non-redundant protein sequence database NCBInr Taxonomy, Bacteria (Eubacteria, https://www.ncbi.nlm.nih.gov/genome/microbes/ ) to identify proteins. The search parameters were set as follows: taxonomy, Viridiplantae (Green Plants; released June 2013; 1437595 sequences, https://www.ncbi.nlm.nih.gov/taxonomy ), trypsin was used as the digestive enzyme, up to one missed cleavage was allowed, carbamidomethylation (Cys) and oxidation (Met) were variable modifications and there were no fixed modifications, peptide tolerance was 50 ppm, and MS/MS tolerance was 0.06 Da. Only significant hits, as defined by the MASCOT probability analysis (p < 0.05), were accepted [49 (link)].
Homology searches (BlastP) of unique sequences and functional annotation by gene ontology terms (GO), InterPro terms (InterProScan, EBI), enzyme classification codes (EC), and metabolic pathways (KEGG, Kyoto Encyclopedia of Genes and Genomes) were performed using the BLAST2GO software suite v2.6.6 (Valencia, Spain).
Homology searches (BlastP) of unique sequences and functional annotation by gene ontology terms (GO), InterPro terms (InterProScan, EBI), enzyme classification codes (EC), and metabolic pathways (KEGG, Kyoto Encyclopedia of Genes and Genomes) were performed using the BLAST2GO software suite v2.6.6 (Valencia, Spain).
6
MALDI-MS Protein Identification Protocol
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MALDI-mass spectrometry (MS) and MALDI-MS/MS were performed on an Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF ion optics (Applied Biosystems, Foster City, CA, USA). Spectra were calibrated externally using five peaks of standard (ABI4700 Calibration Mixture; Applied Biosystems) or internally using porcine trypsin autolysis peptide peaks. In addition to peptide mass finger spectra, the five most abundant pre-cursor ions masses having a signal-to-noise ratio >50 were chosen for MS/MS fragmentation. The interpretation of both the MS and MS/MS data were carried out with the GPS Explorer software (version 3.6; Applied Biosystems). Peaks were extracted from raw spectra by the GPS Explorer software. A combined MS peptide fingerprint and MS/MS peptide sequencing search was performed against the NCBI non-redundant database without taxon restriction using the MASCOT search algorithm. MS/MS peptide spectra with a minimum ion score confidence interval ≥95% were accepted; this was equivalent to a median ion score cut-off of approximately 35 in the data set. Protein identifications were accepted with a statistically significant MASCOT protein search score ≥76 that corresponded to an error probability of P<0.05 in our data set.
7
Comprehensive Analysis of Livestock Performance
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Data for growth performance, meat quality, fatty acid composition, blood variables, diameter and density of adipocytes were analyzed by independent-samples t-test methods using SPSS11.5 (SPSS Inc., Chicago, IL, USA). Results are expressed as mean ±SEM. Data for the scanning two-dimensional gel were analyzed using Image Master 2D image analysis software (PDQuest, Bio-Rad, USA). Proteins were identified using the MASCOT and PROFOUND softwares (http://www.matrixscience.com and http://prowl.rockefeller.edu ) and the GPS Explorer software (version 3.6, Applied Biosystems). Data for Western-blot were calculated by Quantity One software and analyzed by independent-samples t-test methods using SPSS11.5 (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered to be significant and P-values < 0.01 were considered to be highly significant for all the data in this manuscript. In addition, all data and related metadata were deposited in the file at Institute of Animal Science, Guangdong Academy of Agricultural Sciences and available for share.
8
2-DE Protein Identification via MALDI-TOF-MS
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About 500 μg secreted proteins were loaded on the strips, separated by 2-DE and stained with Coomasie Brilliant Blue (CBB) R-250. Differentially expressed protein spots were manually excised from the stained 2-D gel and transferred to a sterile tube (1.5 ml) with 30 % (w/v) Acetonitrile (ACN) and NH4HCO3 (100 mmol) solution to remove the CBB stain. After vacuum drying, the spots were digested in 30 μl enzyme buffer (50 mmol NH4HCO3, 50 ng/μl trypsin (Sigma, USA)) at 37 °C overnight. Then, the small peptides were back extracted using 60 % (w/v) ACN (containing 0.5 % w/v trifluoroacetic acid (TFA)) and dried under a steam of nitrogen. Finally, the peptide samples were re-suspended in 0.8 μl of 50 % (w/v) ACN (containing 0.1 % w/v TFA and 5 mg/ml αcyano-4-hydroxycinnamic acid(CHCA)) and analyzed using a ABI4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, USA). All MALDI-TOF spectra were searched against the National Center for Biotechnology Information non-redundant (NCBInr) database using the GPS Explorer™ software (v3.6, Applied Biosystems) and MASCOT search program (v2.1 Matrix Science). Finally, based on the MALDI-TOF-MS, only protein scores > 95 (p <0.05) were accepted for the identification of protein samples.
9
Protein Identification via MALDI-TOF/TOF
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Protein spots were cut and destained from preparative gels. Mass spectrography (MS) and MS/MS spectra were obtained using the ABI 4800 Proteomics Analyzer Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF/TOF) (Applied Biosystems, Foster City, CA), which operates in a result-dependent acquisition mode. The GPS Explorer software (Applied Biosystems, Foster City, CA) and MASCOT (Matrix Science, London, UK) were utilized with the following parameter: trypsin cleavage, one missed cleavage allowed; carbamido methylation, fixed modification; oxidation of methionines, variable modification; peptide mass tolerance, 100 ppm; fragment tolerance, ±0.3 Da. The minimum ion score confidence interval for MS/MS data was set as 95%.
10
HE4 Protein Identification by IP-MALDI-TOF
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OVCAR-3 cell was immunoprecipitated using an anti-HE4 antibody (Santa Cruz, goat, CA) and combined with 30 ul of protein A/G PLUS agarose (Santa Cruz) by rotating for 1 h at 4°C. The eluents were loaded onto SDS-PAGE gel and Coomassie brilliant blue-stained. The expressed bands were excised and processed for in-gel trypsin digestion and subjected to MALDI-TOF-MS analysis. anti-HE4 antibody was replaced by goat IgG (Bioss, China) for negative control. The peptide and proteins were identified from the MS/MS spectra using the MASCOT algorithm (Matrix Science, Boston, MA). Peptide mass fingerprinting was carried out using the MASCOT search engine from GPS Explorer software (Applied Biosystems, Foster City, CA). Mass spectra used for manual denovo sequencing were annotated with the Data Explorer soft-ware (Applied Biosystems).
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