Computational protocol: Optically-controlled bacterial metabolite for cancer therapy

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Protocol publication

[…] Once tumors reached an approximate size of 200 mm3, 4T1 tumor-bearing mice were randomly divided into two groups with three mice in each group. Then, mice were received intravenously injected of 200 μL of [email protected] coli (with a E. coli concentration of 1 × 108 CFU mL−1) and PBS on the first day. Forty-eight hours after the injection, mice were sacrificed and their tumors were collected. Then, collected tumors were weighed, and homogenized in 200 μL TEAB dissolution buffer. The homogenate was further broken by the ultrasonic cell disruptor for 15 min, and centrifuged at 12,840 × g for 20 min. The supernatant was deposited by adding 800 μL cold acetone containing 10 mM DTT and centrifuged at 12,840 × g for 20 min at 4 °C (Optima L-100XP, Beckmen). To break the disulfide bond of protein, the precipitate was collected and mixed with 800 μL cold acetone and heated to 56 °C. The mixture was subsequently centrifuged at 12,840 × g for 20 min at 4 °C and lyophilized to obtain the protein sample. The sample could be stored at −80 °C for further uses. Total protein concentration was measured using the Bradford method. For protein digestion, 2 μg trypsin was added and then incubated overnight at 37 °C. Then, equal volume of 0.1% FA was added for acidize and peptides were purified on Strata-X C18 pillar. The dried peptides power was redissolved with 20 μL 0.5 M TEAB for peptides labeling.The iTRAQ labeling and fractionation was according to our previous method. Briefly, samples were labeled with iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex U.K. Limited). Next, the labeled samples were fractionated using high-performance liquid chromatography (HPLC) system (Thermo DINOEX Ultimate 3000 BioRS) using a Durashell C18, and 12 fractions were collected for further analysis. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS)/MS analysis was conducted with an AB SCIEX nanoLC-MS/MS (Triple TOF 5600 plus) system. Samples were analyzed by using a 90-min gradient from 2–30% (buffer A 0.1% (v/v) formic acid, 5% (v/v) acetonitrile, buffer B 0.1% (v/v) formic acid, 95% (v/v) acetonitrile). MS1 spectra were collected in the range of 350–1500 m/z for 250 ms. The 20 most intense precursors with charge state 2–5 were selected for fragmentation, and MS2 spectra were collected in the range 50–2000 m/z for 100 ms; precursor ions were excluded from reselection for 15 s. Data of all differential proteins were listed in Supplementary Data .ProteinPilot Software v4.5 was used for analyzing original data. For protein identification, the Paragon algorithm, which was integrated into ProteinPilot was employed against Uniprot Mus musculus (86109 items) for database searching. Proteins with at least one unique peptide and unused value more than 1.3 were collected for further analysis. For protein abundance ratios measured using iTRAQ after normalized, we took a 1.5-fold change and P-value <0.05 as the threshold to identify significant changes. All differential proteins were listed in Supplementary Data .To determine the biological and functional properties of all the identified proteins, identified protein sequences were mapped with Gene Ontology Terms (http://geneontology.org/). For this, homology search was first performed for all the identified sequences with a localized NCBI blast program against NCBI animal database. The e-value was set to <1 × 10-5, and the best hit for each query sequence was taken account for GO term matching. The GO term matching was performed with blast2go v4.5 pipeline5. Clusters of Orthologous Groups of Proteins System (COG, http://www.ncbi.nlm.nih.gov/COG/) was employed for the functional annotation of genes from new genomes and for research into genome evolution. To identify candidate biomarkers, we employed hypergeometric test to perform GO enrichment and KEGG pathway enrichment. To point out the protein–protein interactions, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was employed (http://www.string-db.org/). All other pictures were drawn with R language (http://www.r-project.org/). […]

Pipeline specifications

Software tools BLASTN, Blast2GO
Databases KEGG PATHWAY
Application MS-based targeted proteomics