| Metadata |
| datasetIdentifier | PASS00365 |
| datasetType | MSMS |
| submitter | Adriana Paes Leme <adriana.paesleme@lnbio.cnpem.br> |
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| datasetTag | SCC9orthotopic |
| datasetTitle | ntegrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model |
| publicReleaseDate | 2014-02-01 00:00:00 |
| finalizedDate | 2014-06-16 12:08:04 |
| summary | Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules. |
| contributors | Daniela C. Granato, Mariana R. Zanetti, Rebeca Kawahara, Sami Yokoo, Romênia R. Domingues, Michelle Agostini, Johanna Korvala, Nilva K. Cervigne, Marcelo Falsarella Carazzolle, Ramon Oliveira Vidal, Isadora L. Flores, Alan R. S. Silva, Ricardo D. Coletta, Edgard Graner, Nicholas E. Sherman, Adriana F. Paes Leme |
| publication | PMID: 24858105 |
| growth | The human OSCC cell line SCC-9 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured as recommended. SCC-9 cells are originated from human squamous carcinoma from the tongue. The HaCaT cells, an immortalized but not transformed epithelial cell line [9], was maintained in DMEM containing 10% fetal bovine serum and antibiotics at 37°C in a 5% CO2 air atmosphere. HaCaT cells are human keratinocytes originated from skin. Control cells were used to assure that all the animals were subjected to the same procedures. HaCaT and SCC-9 cells were grown until 75% confluence and 2.5 x 105 cells in 20 μl of phosphate-buffered saline were implanted into the right lateral portion of the tongue of 6- to 8-week-old male Balb/c nude mice, using a syringe with a 30 gauge disposable needle (BD Biosciences). This procedure was approved by the Institutional Committee for Ethics in Animal Research of the University of Campinas. Mice were sacrificed 20 days after implantation and the control and tumor tissues were immediately removed and frozen in dry ice. A small piece of each tumor was fixed in formalin and embedded in paraffin for histopathological examination after H&E staining. We performed three independent experiments for the analysis of the protein and peptide expression in control and tumor tissues. Each sample is composed of a pool of three mouse tissues, either from control or tumor tissues. The samples were named as Control 1 (experiment 1, n=3), Control 2 (experiment 2, n=3), Control 3 (experiment 3, n=3) and Tumor 1 (experiment 1, n=3), Tumor 2 (experiment 2, n=3) and Tumor 3 (experiment 3, n=3). |
| treatment | |
| extraction | The control and tumor tissues were homogenized with liquid nitrogen using mortar and pestle. Tissue protein from each of the three mice were separately resuspended with 50 μl of extraction buffer [10] and incubated at room temperature for 30 min. After centrifugation at 12,000 × g for 10 min at 4°C, the supernatant was quantified using the Bradford method reagent (BioRad) as previously described [11]. Then the same protein amount was pooled from three mouse samples, either from control tissues or from tumor tissues, to be analyzed by LC-MS/MS. Three independent experiments were performed. |
| separation | |
| digestion | The extracted proteins were reduced (5 mM ditiotreitol, 25 min at 56°C), alkylated (14 mM iodoacetamide, 30 min at room temperature in the dark) and digested with trypsin (Promega), the peptides were desalinized using the column Sep-pak C18 cartridge (Waters), dried down in a vacuum concentrator and reconstituted in 0.1% formic acid.
Regarding the identification of endogenous cleavage peptides by LC-MS/MS, 672 μg of extracted protein from tissues as described before were precipitated with the final concentration of 10 mM HCl. After centrifugation, the supernatant was collected, the peptides were desalinized using the column Sep-pak C18 cartridge (Waters) and the peptides were dried down in a vacuum concentrator and resuspended in 20 μl of 0.1% formic acid. |
| acquisition | The protein derived samples (2 μg) and endogenous cleavage peptides were analyzed on an ETD enabled LTQ Velos Orbitrap instrument (Thermo Fisher Scientific) connected to nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) on an EASY-nLC system (Proxeon Biosystem) through a Proxeon nanoelectrospray ion source. The resulting peptides were separated by 2-90% acetonitrile gradient in 0.1% formic acid using a pre-column EASY-Column (2 cm x ID100 μm, 5 μm particle size, Thermo Fisher Scientific) and the a PicoFrit Column (20 cm x ID75 μm, 5 μm particle size, New Objective), at a flow rate 300 nl/min over 135 min. The nanoelectrospray voltage was set to 2.5 kV and the source temperature was 200°C. All instrument methods for the Orbitrap Velos were set up in the data dependent acquisition mode. The full scan MS spectra (from m/z 300-1600) were acquired in the Orbitrap analyzer after accumulation to a target value of 1e6 in the linear ion trap. Resolution in the Orbitrap system was set to r= 60,000 and the 20 most intense peptide ions with charge states ≥ 2 were sequentially isolated to a target value of 10,000 and fragmented in high-pressure linear ion trap by low-energy CID (collision-induced dissociation) normalized collision energy of 35%. The signal threshold for triggering a MS/MS event was set to 1000 counts. Dynamic exclusion was enabled with exclusion size list of 200 and exclusion duration of 60 s. An activation q of 0.25 and activation time of 10 ms were used.For the identification of endogenous cleavage peptides by LC-MS/MS, the samples (4.5 μl) were analyzed on an ETD enabled Orbitrap Velos instrument as described before, except for gradient run that was performed over 45 min. All instrument methods for the LTQ Velos Orbitrap were set up in the data dependent acquisition mode in ETD (electron transfer dissociation), HCD (higher-energy collisional dissociation) and CID fragmentations. For CID fragmentation mode, the same method used for digested proteins was performed. For HCD mode, resolution in the Orbitrap system was set to r= 60,000 and the 5 most intense peptide ions with charge states ≥ 2 were sequentially isolated to a target value of 50,000 and fragmented in HCD with normalized collision energy of 40%, resolution in the Orbitrap system was set to r= 7,500. The signal threshold for triggering a MS/MS event was set to 100,000 counts. Dynamic exclusion was enabled with exclusion size list of 200 and exclusion duration of 20 s and activation time of 10 ms was used. For ETD, resolution in the Orbitrap system was set to r= 60,000 and the 5 most intense peptide ions with charge states ≥ 2 were sequentially isolated to a target value of 50,000 and fragmented in high-pressure linear ion trap and readout in the Orbitrap system with r= 7,500 for MS/MS. The signal threshold for triggering an MS/MS event was set to 500,000 counts. Dynamic exclusion was enabled with exclusion size list of 200 and exclusion duration of 20 s. An activation q of 0.25 and activation time of 100 ms were used, with supplemental activation. |
| informatics | Peak lists (msf) were generated from the raw data files using Proteome Discoverer version 1.3 (Thermo Fisher Scientific) with Sequest search engine and searched against Human and Mouse International Protein Databases (IPI) v. 3.86 (IPI Human: 91,522 sequences; 36,630,302 residues, release July 2011 and IPI Mouse: 58,667 sequences, 26,399,545 residues, release July 2011) with carbamidomethylation as fixed modification, oxidation of methionine as variable modifications, one trypsin missed cleavage and a tolerance of 10 ppm for precursor and 1 Da for fragment ions. The data were analyzed against Human and Mouse databases, considering the orthotopic model, in which the tumor developed in mouse tongue is originated from human cells and the control tissue is originated from mouse tissues.
Regarding the analysis of endogenous cleavage peptides by LC-MS/MS, they were performed as described above, except for the parameters: no enzyme was specified for cleavage and a tolerance of 10 ppm for precursor and 1 Da for fragment ions for top 20 CID (collision-induced dissociation); and for top 5 HCD (higher-energy collisional dissociation) and top 5 ETD (electron-transfer dissociation) fragmentations, a tolerance of 10 ppm for precursor and 0.02 Da for fragment ions were used.
All datasets of proteins and endogenous cleavage peptides were processed using the workflow feature in Proteome Discoverer software and the msf files were analyzed in ScaffoldQ+ v.3.3.2 (Proteome Software), filtered using xcorr cutoffs (+1>1.8, +2>2.2, +3>2.5 and +4>3.5). The scoring parameters in ScaffoldQ+ were set to obtain a false discovery rate less than 1%. |
| instruments | Thermo Scientific LTQ Orbitrap Velos |
| species | Human, mouse |
| massModifications | static: C+57.021464 |
