Computational protocol: Collagen XVI Induces Expression of MMP9 via Modulation of AP-1 Transcription Factors and Facilitates Invasion of Oral Squamous Cell Carcinoma

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Protocol publication

[…] COLXVI cells and mock control cells were synchronized and subsequently cultured in DMEM medium (10% FCS, 1% Penicillin/Streptavidin, 2 mmol/L L-Glutamin) for 24 h. Afterwards, cell lysates were prepared using RIPA buffer. 200 µg of total protein were immunoprecipitated according to the manufacturer’s instructions (Miltenyi Biotec; µMACS™ Protein A/G MicroBeads) with the appropriate antibody. The precipitates were separated by SDS-PAGE. Gels were stained with EZ blue gel staining reagent (Sigma-Aldrich) for 16 h at room temperature. Afterwards, respective bands were excised and gel pieces were washed three times alternately with 50 µL 50 mM NH4HCO3 and 25 mM NH4HCO3 in 50% acetonitrile (Merck, Darmstadt, Germany). Subsequently, the gel slices were dried in a vacuum centrifuge. 5 µL of trypsin solution (Promega, Mannheim, Germany) (12.5 ng/µL in 50 mM ammoniumbicarbonate (AppliChem, Darmstadt, Germany)) were added to each gel piece and incubated at 37°C overnight for in-gel-digestion. The obtained peptides were eluted with 20 µL of 5% formic acid (Merck) and subjected to nano-LC-MS/MS-analysis. Nano-LC was performed on an Ultimate 3000 nano-HPLC-system (Dionex GmbH, Idstein, Germany). Samples were pre-concentrated on a 2 cm x 100 µm I.D. C18-column (nanoseparations, Nieuwkoop, The Netherlands) using 0.1% trifluoroacetic acid (Merck) at a flow-rate of 8 µL/min. The peptides were then separated on a 15 cm×75 µm I.D. C18-PepMap-column (flow-rate 300 µL/min; Dionex GmbH, Idstein, Germany) using a 1 h binary gradient from 5-50% solvent B (solvent A: 0.1% formic acid; solvent B: 0.1% formic acid/84% acetonitrile). The nano-HPLC was coupled directly to a QTOF mass spectrometer (QStar XL, AB Sciex, Darmstadt, Germany) acquiring repeatedly one full-MS and two tandem-MS-spectra of the most intensive ions in the respective full MS-scan. The tandem-MS-spectra were searched against the Uniprot-database using the Mascot Daemon and the Mascot algorithm (version 2.1; Matrix Science Ltd., London, UK) with the following adjustments: taxonomy: Homo sapiens, trypsin as protease, max. one missed cleavage site, oxidation of methionine, pGlu for N-terminal Gln as variable modifications, 0.2 Da-tolerance for MS and MS/MS-signals, only doubly and triply charged ions. Only proteins, for which at least two different peptides could be identified in significantly scored spectra after manual verification, were considered. [...] Significances were determined with assistance of GraphPadPrism V.6 using Mann Whitney U test. P<0.05 was considered as significant. Error bars represent standard deviation. […]

Pipeline specifications

Software tools Mascot Server, GraphPad Prism
Applications Miscellaneous, MS-based untargeted proteomics
Diseases Carcinoma, Squamous Cell