Computational protocol: Expression of both poly r(C) binding protein 1 (PCBP1) and miRNA-3978 is suppressed in peritoneal gastric cancer metastasis

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Protocol publication

[…] Tissue (tumor and adjacent normal) obtained from 5 patients were washed thrice with ice-cold PBS before being lysed using NET buffer (50 mmol/L Tris (pH, 7.4), 150 mmol/L NaCl, 0.1% NP40, 1 mmol/L EDTA, 0.25% gelatin, 0.02% sodium azide) supplemented with protease and phosphatase inhibitor cocktail (ThermoFisher Scientific, Shanghai, China). Lysates were centrifuged at 10,000 rcf for 20 minutes at 4 °C and the supernatant were stored at −80 °C until further use. The lysates were dialyzed against PBS before being reduced and alkylated using 5 mM Tris 2-carboxyethyl phosphine (TCEP) and 10 mM iodoacetamide, respectively. Peptides were desalted, lyophilized, before being re-dissolved in 20 µl of HPLC-grade water containing 0.1% formic acid. Mass spectrometry analysis was performed on a linear ion trap LTQ mass spectrometer (Life Technologies, Shanghai, China) as per manufacturer’s guidelines. Of note, the instrument was operated in positive ion mode and MS full scans were recorded over a mass range of 400–1600 m/z. Post-acquisition of data, ReAdW was used to convert files to mzXML, which were used to query the human IPI database using the Sequest platform. False discovery rate was determined using the Trans-Proteomic Pipeline TPP, with a FDR set at approximately 5%. Finally, relative enrichment or depletion in tumor group was normalized to control group. […]

Pipeline specifications

Software tools ReAdW, Comet
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Stomach Neoplasms