Mascot search program

Manufactured by Matrix Science

Sourced in United Kingdom

MASCOT is a search program used for the identification of proteins from mass spectrometry data. It compares the experimental mass spectra of peptides to theoretical spectra generated from protein sequence databases.

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Lab products found in correlation

Trypsin, by Promega (3 mentions) Trypsin, by Merck Group (2 mentions) Mascot daemon extract msn, by Matrix Science (2 mentions) Mascot 2, by Matrix Science (2 mentions) In gel tryptic digestion kit, by Thermo Fisher Scientific (2 mentions) Sequencing grade modi ed trypsin, by Promega (1 mentions) Ziptip, by Merck Group (1 mentions) Biflex 4 maldi tof ms, by Bruker (1 mentions) Gps explorer software, by Thermo Fisher Scientific (1 mentions) 4700 proteomics analyzer, by Thermo Fisher Scientific (1 mentions) Microflex lrf 20, by Bruker (1 mentions) Oriole fluorescent gel stain, by Bio-Rad (1 mentions) Abi 4800 proteomics analyzer maldi tof tof, by Thermo Fisher Scientific (1 mentions) C18 ziptip, by Merck Group (1 mentions)

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Mascot search engine program MASCOT MS/MS Ion Search program Mascot search MASCOT program Mascot

16 protocols using mascot search program

Identifying Protein Modifications by Mass Spectrometry

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The band of the SDS gel was excised and destained. Trypsin digestion was performed at 37 °C for 4 h (In-Gel Tryptic Digestion Kit, Thermo Fisher Scienti c) in order to identify the peptide sequence by mass spectrometry (MS). Desalting of the tryptic digested peptides were performed on a C18 proteomic column (Mass Solution Ltd., Taipei, Taiwan). MS analysis of the resulting peptides applying nLC/Q-TOF (Micromass, Manchester, UK) was performed. The resulting MS data were used to search against entries in the NCBI database using the MASCOT search program (Matrixscience, London, UK). Additionally, peptides with acetylated lysines were predicted. The parameters searched for were: mass values: monoisotopic; fragment mass tolerance: ± 0.4 Da; protein mass: unrestricted; maximal missed cleavages:
1; peptide mass tolerance: ± 0.4 Da; variable modi cation: oxidation in methionine; acetylation in lysine: carbamidomethylation in cysteine.

Gurunathan R., Huang B., Ponnusamy V.K., Hwang J., & Dahms H. (2020). Novel Recombinant keratin Degrading Subtilisin Like Serine Alkaline Protease from Bacillus Cereus Isolated from Marine Hydrothermal Vent Crabs .

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Protein Identification by Mass Spectrometry

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A total of 1 µg of the protein fraction extracted with 275 mM NaCl in the cation-exchange chromatography was fractionated on a polyacrylamide gel and visualized by Coomassie blue staining. The protein band of molecular mass 37 kDa was cut out and treated with trypsin. The tryptic peptides were separated and analyzed by nano ACQUITY ultra performance liquid chromatography coupled directly to a LTQ-orbitrap-mass spectrometer (LC-MS/MS). The spectra from the LC-MS/MS were processed using the SEQUEST (Thermo Quest, San Jose, CA, USA) software and the peak list files generated were used to query the National Center for Biotechnology Information using the MASCOT search program (Matrix Science Ltd., London, UK, http://www.matrixscience.com/).

Xu M.L., Kim H.J., Kim S.C., Ju W., Kim Y.H., Chang K.H, & Kim H.J. (2019). Serum anti-GAPDH autoantibody levels reflect the severity of cervical lesions: A potential serum biomarker for cervical cancer screening. Oncology Letters, 18(1), 255-264.

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Proteomic analysis of mt-nucleoid

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The pelleted of different stages mt-nucleoid were washed with25mM NH 4 HCO 3 solution for 20 min, and then dried in a vacuum centrifuge. Resuspended the protein 50μl 25 mM NH 4 HCO 3 solution with 12.5 ng/μl trypsin (Sequencing Grade Modi ed Trypsin, Promega), and incubated for 12 to 16 h at 37℃. An 50μl solution containing 5% (w/v) formic acid and 50% (w/v) acetonitrile was added to the pelleted, agitated for 1 h, and moved into a new tube. Then repeated with solution containing 2.5% (w/v) formic acid and 50% (w/v) acetonitrile for 1 h to to recover the tryptic peptides.
The tryptic peptides were puri ed with a PepClean C-18 spin column (Pierce). Pellets of tryptic peptides were dissolved in 10 to 20 μl of 0.1% (w/v) formic acid for LC-MS/MS analysis. Data were analyzed with an in-house Mascot search program (v. 2.2.06; Matrix Science) against the UniProtKB/Swiss-Prot Viridiplantae database (green plants, 28,773 protein entries). The database search for protein identi cation was performed as described (Lo et al. 2011) . All stages of data are from duplicate studies.

Cheng N., Lo Y., Lin N., & Dai H. (2022). Dynamics of mitochondrial nucleoid proteins involved in development of cotyledon mitochondria during mung bean seed germination.

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Protein Identification by MALDI-TOF Mass Spectrometry

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Samples were separated by SDS–PAGE and stained with Coomassie blue. Gel slices containing the proteins to be identified by mass spectrometry were treated with trypsin (Sigma) for 12 h at 37°C. The peptides were extracted from the gel slices using 0.1% trifluoroacetic acid and 50% acetonitrile and then concentrated using vacuum centrifugation. The resulting peptide solution was deionized using a ZipTip (Millipore), mixed with a matrix solution (10 mg/mL α-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid, and 50% acetonitrile), and dried on a stainless steel plate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) was conducted using an ultraflex TOF/TOF (Burker Daltonics). Peptide mass fingerprinting was performed using the Mascot search program (Matrix Science).

Yu M., Sun L., Zhang Z., Zhang Y., Zhang H., Na L, & Wang X. (2022). KPNA6 is a Cofactor of ANP32A/B in Supporting Influenza Virus Polymerase Activity. Microbiology Spectrum, 10(1), e02073-21.

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Protein Identification Using Mascot

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Peak lists of proteins were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo, London, UK) using the default parameters, and submitted to Mascot 2.1 (Matrix Science). All MS/MS spectra of differentiating peptides were searched against human non-redundant NCBInr database using the Mascot search program (Matrix Science, London, UK, www.matrixscience.com) for protein identification with the following criteria: (1) species, Homo sapiens; (2) allowed one missed cleavage; (3) variable modifications, Oxidation (M), Phospho (ST) and Phospho (Y); (4) peptide tolerance, ±6 ppm; (5) MS/MS tolerance, ± 0.6Da; (6) peptide +2, +3 and +4; and (7) enzyme specificity, none. The results were imported into Progenesis QI LC-MS software and peptides were considered to be confidently identified when matches had a high ion score >20 and peptides were assigned to a protein.

Hao J., Graham P., Chang L., Ni J., Wasinger V., Beretov J., Deng J., Duan W., Bucci J., Malouf D., Gillatt D, & Li Y. (2016). Proteomic identification of the lactate dehydrogenase A in a radioresistant prostate cancer xenograft mouse model for improving radiotherapy. Oncotarget, 7(45), 74269-74285.

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MALDI-TOF-MS Protein Identification Protocol

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Samples were air-dried and analyzed by a Biflex IV MALDI-TOF-MS (Bruker, Billerica, MA, USA). The N₂ laser was operated at an accelerating voltage of 19 kV with a wavelength of 337 nm (3 ns pulse length).
Data analysis was performed with the National Center for Biotechnology Information (NCBI) nr database using the MASCOT search program (Matrix Science, Boston, MA, USA). The following parameters were allowed: taxonomy restrictions to other firmicutes, 120 ppm mass tolerance in MS, one missed cleavage, oxidation (M) as a variable modification and carbamidomethyl (C) as a fixed modification. The confidence in the peptide mass fingerprinting (PMF) matches (p < 0.05) was based on the MOWSE score and confirmed by the accurate overlapping of the matched peptides with the major peaks of the mass spectrum. Only the best matches with high confidence levels were chosen when the software gave more than one eligible result.

Zhao J., Zhang C, & Lu Z. (2018). Differential proteomics research of Bacillus amyloliquefaciens and its genome-shuffled saltant for improving fengycin production. Brazilian Journal of Microbiology, 49(Suppl 1), 166-177.

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Proteomic Analysis of FLAG-ADD1 Interactome

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FLAG-ADD1 was transiently overexpressed in HEK293 cells, and the cells were synchronized in the G2/M phase by nocodazole. FLAG-ADD1 was bound to anti-FLAG M2 affinity gel and was eluted by 100 µM 3× FLAG peptides. FLAG-ADD1 and its interacting proteins were fractionated by SDS-PAGE and were stained with Coomassie blue. MS for the identification of protein IDs and phosphorylation sites was performed as previously described (Chan et al., 2010 (link)). In brief, nanoscale capillary liquid chromatography (LC)/tandem MS (LC-MS/MS) analysis was performed using an LC system (UltiMate Capillary; LC Packings) coupled to a quadrupole time-of-flight mass spectrometer (QSTAR; Applied Biosystems). A nanoelectrospray interface was used for LC-MS/MS analysis. Ionization (2.0-kV ionization potential) was performed with a coated nano-LC tip. Data acquisition was performed by automatic Information Dependent Acquisition (Applied Biosystems). The Information Dependent Acquisition automatically finds the most intense ions in a time-of-flight MS spectrum and then performs an optimized MS/MS analysis on the selected ions. The product ion spectra generated by nano–LC-MS/MS were searched against NCBI databases for exact matches using the MASCOT search program (Matrix Science).

Chan P.C., Hsu R.Y., Liu C.W., Lai C.C, & Chen H.C. (2014). Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X. The Journal of Cell Biology, 204(1), 19-28.

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Protein Identification by Mass Spectrometry

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The band of the SDS gel was excised and destained. Trypsin digestion was performed at 37 °C for 4 h (In-Gel Tryptic Digestion Kit, Thermo Fisher Scientific) in order to identify the peptide sequence by mass spectrometry (MS). Desalting of the tryptic digested peptides were performed on a C18 proteomic column (Mass Solution Ltd., Taipei, Taiwan). MS analysis of the resulting peptides applying nLC/Q-TOF (Micromass, Manchester, UK) was performed. The resulting MS data were used to search against entries in the NCBI database using the MASCOT search program (Matrixscience, London, UK). Additionally, peptides with acetylated lysines were predicted. The parameters searched for were: mass values: monoisotopic; fragment mass tolerance: ± 0.4 Da; protein mass: unrestricted; maximal missed cleavages: 1; peptide mass tolerance: ± 0.4 Da; variable modification: oxidation in methionine; acetylation in lysine: carbamidomethylation in cysteine.

Gurunathan R., Huang B., Ponnusamy V.K., Hwang J.S, & Dahms H.U. (2021). Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs. Scientific Reports, 11, 12007.

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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.

Chen X., Deng Z., Yu C., Yan C, & Chen J. (2016). Secretome analysis of rice suspension-cultured cells infected by Xanthomonas oryzae pv.oryza (Xoo). Proteome Science, 14, 2.

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Protein Identification using Mascot Search

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Peak lists of proteins were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo, London, UK) using the default parameters, and submitted to Mascot 2.1 (Matrix Science). All MS/MS spectra of differentiating peptides were searched against human non-redundant NCBInr database using the Mascot search program (Matrix Science, London, UK, www.matrixscience.com) for protein identification with the following criteria: (1) species, Homo sapiens; (2) allowed one missed cleavage; (3) variable modifications, Oxidation (M), Phospho (ST) and Phospho (Y); (4) peptide tolerance, ±6 ppm; (5) MS/MS tolerance, ± 0.6 Da; (6) peptide +2, +3 and +4; and (7) enzyme specificity, none. The results were imported into Progenesis LC-MS software and peptides were considered to be confidently identified when matches had a high ion score >20 and peptides were assigned to a protein.

Chang L., Ni J., Beretov J., Wasinger V.C., Hao J., Bucci J., Malouf D., Gillatt D., Graham P.H, & Li Y. (2017). Identification of protein biomarkers and signaling pathways associated with prostate cancer radioresistance using label-free LC-MS/MS proteomic approach. Scientific Reports, 7, 41834.

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