Proteome discoverer 2

Manufactured by Matrix Science

Sourced in United Kingdom

Proteome Discoverer 2.2 is a software solution for the analysis and identification of proteins from mass spectrometry data. It provides a comprehensive platform for processing, analyzing, and managing proteomic data.

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20 protocols using proteome discoverer 2

Quantitative Proteome and Phosphoproteome Analysis

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The resulting MS/MS data were processed using the MASCOT engine (Matrix Science, London, UK; version 2.2) embedded into Proteome Discoverer 2.4. The data were searched against the database “Homo_sapiens_194324” and against a library of common protein contaminants (for filtering out contaminant proteins), and an anti-database was added to assess the false discovery rate (FDR) due to random matches. The following system parameters were applied: restriction enzyme digestion method, trypsin/P; number of missed cleavage sites, 2; peptide mass tolerance, ± 20.0 ppm; fragment mass tolerance, 0.1 Da; fixed modification, carbamidomethyl (C); variable modifications, “Oxidation (M)”, “Phospho(ST)”, “Phosp (Y)”; and FDR, 1%.
Only proteins whose levels differed > 2-fold or < 0.83-fold between cultures treated with triptolide or vehicle (in association with p < 0.05) were considered in subsequent bioinformatics analyses. A similar criterion was applied to select phosphorylation sites in the proteome.

Song X., He H., Zhang Y., Fan J, & Wang L. (2022). Mechanisms of action of triptolide against colorectal cancer: insights from proteomic and phosphoproteomic analyses. Aging (Albany NY), 14(7), 3084-3104.

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Characterization of PI3K Interactome

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Recombinant PI3K (10 μg/ml) was incubated with 20 μM MVK for 10 min at room temperature (RT). The protein mixture was reduced and carboxymethylated to remove the S–S bond. After SDS–PAGE and Coomassie brilliant blue staining, the bands were excised and digested using trypsin or AspN. The digestion mixture was subjected to Q Exactive mass spectrometry (MS). MS and tandem MS data were acquired using the data-dependent top10 method. The resulting tandem MS data were searched by MASCOT 2.7 (https://www.matrixscience.com/) and quantified using Proteome Discoverer2.4 (proteome-discoverer-software.html">https://www.thermofisher.com/jp/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html) with variable modifications. In Table S2, the MVK modification site is indicated as a CRA modification because CRA modification is registered in the database with the exact same composition as MVK modification. The residues that were more than 1% bound to MVK compared with the control were designated MVK target residues (Table S3).

Morimoto A., Takasugi N., Pan Y., Kubota S., Dohmae N., Abiko Y., Uchida K., Kumagai Y, & Uehara T. (2024). Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling. The Journal of Biological Chemistry, 300(3), 105679.

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Quantitative Proteome Data Analysis

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The MS raw data was identified and quantified using Proteome Discoverer 2.4 software, including the MASCOT engine (Matrix Science, London, UK; version 2.2). The parameters were as follows: ±20 ppm for peptide mass tolerance, 0.1 Da for fragment mass tolerance, Decoy for database pattern, and a peptide false discovery rate (FDR) of less than or equal to 0.01.

Yu H., Ning N., He F., Xu J., Zhao H., Duan S, & Zhao Y. (2024). Targeted Delivery of Geraniol via Hyaluronic Acid-Conjugation Enhances Its Anti-Tumor Activity Against Prostate Cancer. International Journal of Nanomedicine, 19, 155-169.

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Comprehensive Proteomic Analysis with iTRAQ

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MS/MS spectra were searched using the MASCOT engine (Matrix Science; version 2.2) embedded into Proteome Discoverer 2.4. The following parameters were used: enzyme: trypsin; max missed cleavages: 2; fixed modifications: carbamidomethyl (C), iTRAQ4/8 plex (N‐term), iTRAQ 4/8 plex (K) or TMT 6/10/16 plex (N‐term), iTRAQ6/10/16 plex (K); variable modifications: oxidation (M)/phospho(ST)/phospho(Y); database: Swissprot_Mus_musculus_17063_20210106.fasta; peptide mass tolerance: ±20 ppm; fragment mass tolerance: 0.1 Da; peptide FDR: ≤0.01; and protein FDR: ≤0.01.

Bai Y., Li L., Dong B., Ma W., Chen H, & Yu Y. (2022). Phosphorylation‐mediated PI3K‐Art signalling pathway as a therapeutic mechanism in the hydrogen‐induced alleviation of brain injury in septic mice. Journal of Cellular and Molecular Medicine, 26(22), 5713-5727.

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Mascot-based Proteome Profiling

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MS/MS raw files were processed using MASCOT engine (Matrix Science, London, UK; version 2.6) embedded into Proteome Discoverer 2.2, and searched against the database (Uniprot_HomoSapiens_20386_20180905). The search parameters were as follows: trypsin as the enzyme with a maximum of two missed cleavages permitted. The mass tolerance was set to 10 ppm for precursor ions and 0.05 Da for MS2 fragments. Carbamidomethyl of Cysteine was set as fixed modification and Oxidation (M) and Acetyl (Protein N-term) were specified as dynamic modifications.

Zhu S., Fu W., Hu J., Tang X., Cui Y, & Jia W. (2022). Quantitative proteomics reveals specific protein regulation of severe hypospadias. Translational Andrology and Urology, 11(4), 495-508.

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Quantitative Proteomic Analysis of Salt Stress Response

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GXDK6 was incubated for 16 h under 0, 5, and 10% NaCl stress, respectively. The cells were collected by centrifugation at 8,000 × g for 10 min. The total proteins in GXDK6 were extracted by SDT lysis methods (Zhu et al., 2014 (link)). Proteome sequencing and analysis of the extracted proteins were conducted using a tandem mass tag (TMT)-based quantitative proteomics (Myers et al., 2018 (link)). TMT-labeled peptides were fractionated by RP chromatography using Agilent 1260 infinity II HPLC. LC–MS/MS analysis was performed on a Q Exactive plus mass spectrometer (Thermo Fisher Scientific) that was coupled to Easy nLC (Thermo Fisher Scientific) for 60/90 min. The MS/MS raw files were processed using MASCOT engine (Matrix Science, London, United Kingdom; version 2.6) embedded into Proteome Discoverer 2.2, and searched against the NCBI/NR/UniProt database. Proteins with fold change>1.2 and p value (Student’s t test) <0.05 were considered as differentially expressed proteins (DEPs).

Cai X., Sun H., Yan B., Bai H., Zhou X., Shen P, & Jiang C. (2023). Salt stress perception and metabolic regulation network analysis of a marine probiotic Meyerozyma guilliermondii GXDK6. Frontiers in Microbiology, 14, 1193352.

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Proteomic Analysis of Peptide 3 Treatment in H358 Cells

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H358 cells were treated with 20 μmol/L of peptide 3 or with equal volume of PBS as control, and were incubated in a humidified atmosphere of 5% CO₂ at 37 °C for 24 h. Cell samples were collected and then handed over to LC-Bio Technology Co., Ltd. for the subsequent proteome analysis with TMT technology. Quantitative proteome analysis involved the following six steps in turn: sample preparation, SDS-PAGE separation, filter-aided sample preparation (FASP Digestion), TMT labeling, peptide fractionation with reversed phase chromatography, and mass spectrometry analysis. MS/MS raw files were processed using MASCOT engine (Matrix Science, London, UK; version 2.6) embedded into Proteome Discoverer 2.2, and searched against the Protein Database for peptide identification. A peptide and protein false discovery rate of 1% was enforced using a reverse database search strategy. Proteins with Fold change >1.2 and P value (Student's t-test) < 0.05 were considered to be differentially expressed proteins, which were subjected to GO and KEGG analysis.

Li C., Zhao N., An L., Dai Z., Chen X., Yang F., You Q., Di B., Hu C, & Xu L. (2021). Apoptosis-inducing activity of synthetic hydrocarbon-stapled peptides in H358 cancer cells expressing KRASG12C. Acta Pharmaceutica Sinica. B, 11(9), 2670-2684.

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Differential Proteomics Analysis Pipeline

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MS/MS raw files were processed using MASCOT engine (Matrix Science, London, United Kingdom; version 2.6) embedded into Proteome Discoverer 2.2, and searched against the NCBI database (Accession: NZ_CP006961.1 and NZ_CP006962.1). The search parameters included trypsin as the enzyme used to generate peptides with a maximum of two missed cleavages permitted. A precursor mass tolerance of 10 ppm was specified and 0.05 Da tolerance for MS2 fragments. Except for TMT labels, carbamidomethyl (C) was set as a fixed modification. Variable modifications were Oxidation (M) and Acetyl (Protein N-term). A peptide and protein false discovery rate of 1% was enforced using a reverse database search strategy. Proteins with fold change (FC) >1.5, p-value < 0.05 (by Student’s t-test), and the false discovery rate (FDR) < 0.05 were considered differentially expressed.

Zhang G., Zhong F., Chen L., Qin P., Li J., Zhi F., Tian L., Zhou D., Lin P., Chen H., Tang K., Liu W., Jin Y, & Wang A. (2021). Integrated Proteomic and Transcriptomic Analyses Reveal the Roles of Brucella Homolog of BAX Inhibitor 1 in Cell Division and Membrane Homeostasis of Brucella suis S2. Frontiers in Microbiology, 12, 632095.

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Proteome Discoverer 2.2 MS/MS Analysis

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The MASCOT engine was built into Proteome Discoverer 2.2 (Matrix Science, London, UK; 2.6), which processes raw MS/MS documents and retrieves them from UniProt-Homo_sapiens.fasta. The search parameters included trypsin-generated peptides and allowed a maximum of 2 missed cleavages. A precursor mass of 10 ppm and an MS2 fragment of 0.05 Da were specified. The amino acid (C) sequence was set as a fixed modification in addition to the TMT tag. The variable modifications were (M)oxidation and acetylation (N-protein). A reverse database search strategy was used to increase the detection rate of peptides and proteins by 1% (fold variation of 1.2, p value determined by Student’s t-test).

Fu Y., Huang X.Q., Qu H.B., Ge Y.Z, & Ru X.L. (2024). Tandem Mass Tag-Based Proteomic Analysis of Normal and Degenerated Human Intervertebral Discs. Journal of Pain Research, 17, 1313-1326.

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Quantitative Histone PTM Analysis

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Database search was performed in Proteome Discoverer 2.2 using the Mascot (v2.5, Matrix Science) search engine. Spectra were deconvoluted with Xtract and search against human histones H3 (UniProt). Enzyme was set to Glu-C with 0 missed cleavages allowed. Variable modifications were acetylation (K), mono- and di-methylation (KR), tri-methylation (K) and phosphorylation (STY). Mass tolerance was set 2.1 Da for precursor and 0.01 for fragment ions.
Mascot output files were exported as CSV files and processed with our in-house software ProteoformQuant for confident identification and quantification [32 (link)]. Peptides without sufficient fragment ions to unambiguously localize PTM were automatically discarded by the software. Co-eluting peptides were assigned abundances based on the ratio of unique fragments. The relative abundance of a given modified peptide was calculated by dividing the total ion intensity of this peptide by the sum of all peptides sharing the same sequence.

Lu C., Coradin M., Janssen K.A., Sidoli S, & Garcia B.A. (2021). Combinatorial histone H3 modifications are dynamically altered in distinct cell cycle phases. Journal of the American Society for Mass Spectrometry, 32(6), 1300-1311.

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