Mascot server v2

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Mascot Server v2.5.1 is a software application that provides a platform for protein identification and characterization from mass spectrometry data. The software is designed to analyze and interpret the results of mass spectrometry experiments, enabling researchers to identify and quantify proteins within complex biological samples.

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24 protocols using mascot server v2

Protein Identification by Mass Spectrometry

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The mass spectrometry data obtained were processed using PeakView v2.2 Software (SCIEX) and exported as mgf files, which were searched using Mascot Server v2.6.0 (Matrix Science, London, UK) against Homo sapiens protein database from Uniprot (last update: 20170331, 141.978 sequences), together with commonly occurring contaminants.
Search parameters were set as follows: enzyme, trypsin; allowed missed cleavages, 2; carbamidomethyl (C) as fixed modification and acetyl (Protein N-term), pyrrolidone from E, pyrrolidone from Q and Oxidation (M) as variable modifications. Peptide mass tolerance was set to ±25 ppm for precursors and 0.05 Da for fragment masses. The confidence interval for protein identification was set to ≥95% (p < 0.05) and only peptides with an individual ion score above the 1% false discovery rates (FDR) at spectra level were considered correctly identified.

Lozano V., Martín C., Blanco N., Alcalde I., Fernandez-Vega Cueto L., Merayo-Lloves J, & Quirós L.M. (2022). Exosomes Released by Corneal Stromal Cells Show Molecular Alterations in Keratoconus Patients and Induce Different Cellular Behavior. Biomedicines, 10(10), 2348.

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Peptide Identification by MASCOT MS/MS

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Mascot server v2.6.0 (www.matrix-science.com, UK) in MS/MS ion search mode (local licenses) was applied to conduct peptide matches (peptide masses and sequence tags) and protein searches against Swiss Protein v2017_02 (553655 sequences; 198777566 residues) using all entries as well as a taxonomy filter for Human (Homo sapiens) (20169 sequences). The following parameters were set for the search: carbamidomethyl (C) on cysteine was set as fixed; variable modifications included asparagine and glutamine deamidation, methionine oxidation, Hg (I) at cysteine as well mercury Hg (II) at cysteine and methionine. Two missed cleavages were allowed; monoisotopic masses were counted; the precursor peptide and fragment mass tolerance were set at 20 ppm and 0.3 Da respectively for the data coming from the Q-TOF, and precursor and fragment masses of 15 ppm and 0.02 Da for QExactive Plus Orbitrap FTMS; the ion score or expected cut-off was set at 5. The MS/MS spectra were searched with MASCOT using a 95% confidence interval (% C.I.) threshold (p<0.05), with which a minimum score of 36 was used for peptide identification. The protein redundancy that appeared at the database under different gi and accession numbers was limited to Human. All of the proteins identified in the current study were found in these domains.

Maniero M.Á., Wuilloud R.G., Callegari E.A., Smichowski P.N, & Fanelli M.A. (2019). Metalloproteomics analysis in human mammary cell lines treated with inorganic mercury. Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS), 58, 126441.

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Brassicaceae Proteome Mass Spectrometry

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Mass spectrometry data obtained were processed using PeakView v2.2 Software (SCIEX) and exported as mgf files which were found using Mascot Server v2.6.0 (Matrix Science, London, UK) against the Brassicaceae protein database from Uniprot (last update: 20181026, 483.295 sequences), together with commonly occurring contaminants. Search parameters were set as follows: Enzyme, trypsin; allowed missed cleavages, 2; carbamidomethyl (C) as fixed modification and acetyl (Protein N-term), pyrrolidone from E, pyrrolidone from Q and oxidation (M) as variable modifications. Peptide mass tolerance was set to ±25 ppm for precursors and 0.05 Da for fragment masses. The confidence interval for protein identification was set to ≥95% (p < 0.05) and only peptides with an individual ion score above 20 were considered correctly identified. Predictions of transmembrane helices (TMH) (Table S5) were performed with TMHMM Server v. 2.0 [82 (link)], available at http://www.cbs.dtu.dk/services/TMHMM/. Predictions of protein location were performed with DeepLoc-1.0 [83 (link)] and UniProtKB [84 (link)].

Yepes-Molina L., Carvajal M, & Martínez-Ballesta M.C. (2020). Detergent Resistant Membrane Domains in Broccoli Plasma Membrane Associated to the Response to Salinity Stress. International Journal of Molecular Sciences, 21(20), 7694.

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Proteomic Analysis of Human Samples

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Samples were shipped at −80 °C to the Proteomics Facility of CEINGE-Biotecnologie Avanzate (Naples, Italy) for LC–MS/MS, which was performed on a Proxeon EASY nano liquid chromatography system coupled with an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
Searches for matches between the MS data and proteins in the NCBI nr and Swiss-Prot databases (human taxonomy) were performed with the Mascot Server v2.3 (Matrix Science, Boston, MA, USA). Searches were conducted with carbamidomethylation of cysteine as a fixed modification; oxidation of methionine, pyroglutamate formation from glutamine and pyro-carbamidomethyl as variable modifications of proteins; a peptide mass tolerance of ±10 ppm; a fragment mass tolerance of ±0.6 Da; and an allowance for up to two missed tryptic cleavages.

Repetto O., Lovisa F., Elia C., Enderle D., Romanato F., Buffardi S., Sala A., Pillon M., Steffan A., Burnelli R., Mussolin L., Mascarin M, & De Re V. (2021). Proteomic Exploration of Plasma Exosomes and Other Small Extracellular Vesicles in Pediatric Hodgkin Lymphoma: A Potential Source of Biomarkers for Relapse Occurrence. Diagnostics, 11(6), 917.

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Protein Identification by 2D-DIGE and LC-MS/MS

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To identify the proteins of the differentially abundant spots, a 2D-DIGE pick gel was prepared and separated. After Coomassie blue staining, spots of interest were excised with the Screen Picker (Proteomics Consult), destained overnight with 25 mM ammonium bicarbonate in 50% acetonitrile, dehydrated with 100% acetonitrile, dried, and digested overnight with trypsin (200 ng/spot; T6567 Sigma-Aldrich) in 40 mM ammonium bicarbonate, 9% acetonitrile at 37 °C. The trypsin digest was extracted with 1% trifluoroacetic acid (Sigma-Aldrich), lyophilized and stored at − 80 °C until shipping.
Samples were analyzed at the Proteomics Facility of CEINGE-Biotecnologie Avanzate (Naples, Italy). LC–MS/MS was done on a Proxeon EASY nano liquid chromatography system coupled with an LTQ Orbitrap XL mass spectrometer with ETD (Thermo Fisher Scientific, Massachusetts, USA). CEINGE provided raw MS data in.mgf format.
Mascot Server v2.3 (Matrix Science, Boston, USA) was used to search for matches between the MS data and proteins in the NCBI nr and Swiss-Prot databases, selected for human taxonomy.

Repetto O., De Re V., Giuffrida P., Lenti M.V., Magris R., Venerito M., Steffan A., Di Sabatino A, & Cannizzaro R. (2021). Proteomics signature of autoimmune atrophic gastritis: towards a link with gastric cancer. Gastric Cancer, 24(3), 666-679.

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Phosphoproteomics of EGF-treated PFKP

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An in vitro KAT5-acetylated purified PFKP was digested in-gel in 50 mM ammonium bicarbonate buffer containing Rapigest (Waters Corp., Milford, MA) overnight at 37°C with 200 ng of sequencing-grade modified trypsin (Promega, Madison, WI). The digest was analyzed by LC-MS/MS on an Orbitrap-Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA).
Immunoprecipitated PFKP protein from EGF-treated U251 cells was digested and analyzed by LC-MS/MS on an Orbitrap-Ellite mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Proteins were identified by searching of the fragment spectra against the SwissProt protein database (EBI) using Mascot Server v.2.3 (Matrix Science, London, UK) and SEQUEST v.1.27 (University of Washington, Seattle, WA) via Proteome Discoverer software v.1.4 (Thermo Fisher Scientific). Phosphopeptide matches were analyzed by using the PhosphoRS algorithm implemented in Proteome Discoverer and manually curated (Taus et al., 2011 (link)).

Lee J.H., Liu R., Li J., Wang Y., Tan L., Li X.J., Qian X., Zhang C., Xia Y., Xu D., Guo W., Ding Z., Du L., Zheng Y., Chen Q., Lorenzi P.L., Mills G.B., Jiang T, & Lu Z. (2018). EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation. Molecular cell, 70(2), 197-210.e7.

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Protein Identification via HPP Guidelines

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The selected
data sets were analyzed for protein identification following the HPP
guidelines. We searched all the mgf files downloaded from PRIDE against
the neXtProt database (release 20150901) using the target-decoy strategy
with an in-house Mascot Server v. 2.3 (Matrix Science, London, U.K.)
search engine. A decoy database was created using the peptide pseudoreversed
method, and separate searches were performed for target and decoy
databases.
For each sample, searching parameters were fixed
on the basis of the information provided in the metadata associated
with the project in PRIDE or by the methods described in the referenced
article. False Discovery Rates at the PSM level and protein level
using Mayu^{24 (link)} were calculated, and protein
identifications were obtained applying the criteria of PSM FDR <
1% and protein FDR < 1%. Protein inference was performed using
the PAnalyzer algorithm.^{25 (link)} Only those missing
proteins labeled as conclusive by this algorithm and with at least
2 proteotypic peptides were considered as observed missing proteins
in the sample.

Garin-Muga A., Odriozola L., Martínez-Val A., del Toro N., Martínez R., Molina M., Cantero L., Rivera R., Garrido N., Dominguez F., Sanchez del Pino M.M., Vizcaíno J.A., Corrales F.J, & Segura V. (2016). Detection of Missing Proteins Using the PRIDE Database as a Source of Mass Spectrometry Evidence. Journal of Proteome Research, 15(11), 4101-4115.

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Mass Spectrometry Proteomics Workflow

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Mass spectrometry was performed with the UltiMate 3000 Nano LC system, Q-Exactive (Thermo Fisher Scientific) and analyzed with MASCOT software (Mascot Distiller v2.5, Mascot Server v2.5, www.matrixscience.com) (Matrix Science, London, UK) by CoMIT Omics Center in Osaka University.

Kojima-Kita K., Kuramochi-Miyagawa S., Nakayama M., Miyata H., Jacobsen S.E., Ikawa M., Koseki H, & Nakano T. (2021). MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells. Scientific Reports, 11, 20472.

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Proteomic Analysis of Peptide-Induced Pulpal Wound Healing

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To investigate the mechanism by which specific peptides promoted pulpal wound healing, liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed to quantify and compare protein expression patterns in hDPSCs. The hDPSCs (3 million cells per 100-mm dish) were cultured with 0.1 mM of functional peptide No. 1 or recombinant S100A8 (ProSpec) in mineralization induction medium for 7 d. Proteins were extracted from the cell membrane or cytoplasm using a Cell Membrane Protein Extraction Kit (Cosmo Bio). The hDPSCs cultured in the same medium supplemented with PBS were used as a control group.
Samples were processed using trypsin and lysyl endopeptidase. Ionization was performed using the UltiMate 3000 Nano LC system (Thermo Fisher Scientific) through an ESI column (0.075 × 150 mm). The mobile phase was a 5% acetonitrile solution that contained 0.1% formic acid; the flow rate was 300 nL/min. The acquired MS spectra were analyzed via Q-Exactive software (Thermo Fisher Scientific). Potential proteins were identified and quantified using peptide mass fingerprinting software (Mascot Distiller and Mascot Server v2.5; Matrix Science), based on UniProt data. Proteins identified by MS/MS were quantitatively compared using Scaffold Viewer (Proteome Software) and refined in accordance with the criteria shown in Appendix Table 2.

Watanabe M., Okamoto M., Komichi S., Huang H., Matsumoto S., Moriyama K., Ohshima J., Abe S., Morita M., Ali M., Takebe K., Kozaki I., Fujimoto A., Kanie K., Kato R., Uto K., Ebara M., Yamawaki-Ogata A., Narita Y., Takahashi Y, & Hayashi M. (2022). Novel Functional Peptide for Next-Generation Vital Pulp Therapy. Journal of Dental Research, 102(3), 322-330.

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Proteomic Profiling of IFNAR1 Knockout in BECs

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Acquired raw data files were analyzed using Proteome Discoverer v2.5 (Thermo Fisher Scientific) with Mascot Server v2.5.1 (Matrix Science Ltd., UK) search engine against mouse protein database (SwissProt). A maximum of two missed cleavage sites were allowed for full tryptic digestion, while setting the precursor and the fragment ion mass tolerance to 10 ppm and 0.02 Da, respectively. Carbamidomethylation of cysteine was specified as a fixed modification, while TMT6plex on lysine and N-termini, oxidation on methionine as well as deamidation of asparagine and glutamine were set as dynamic modifications. Initial search results were filtered with 5% FDR using the Percolator node in Proteome Discoverer. Quantification was based on the TMT-reporter ion intensities. Proteome discrimination of three samples per genotype (IFNAR1 KO or wild-type) was obtained using all proteome features with high-dimensional analysis methods; principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Volcano plots were used to identify proteome features differentially represented in IFNAR1 KO BEC samples.

Shafi A.M., Végvári Á., Zubarev R.A, & Penha-Gonçalves C. (2023). Brain endothelial cells exposure to malaria parasites links type I interferon signalling to antigen presentation, immunoproteasome activation, endothelium disruption, and cellular metabolism. Frontiers in Immunology, 14, 1149107.

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