DATASET S2: MS/MS raw data from Pollicipes pollicipes Cement Gland
Data for the article: Domínguez-Pérez, D.; Almeida, D.; Wissing, J.; Machado, A.M.; Jänsch, L.; Castro, L.F.; Antunes, A.; Vasconcelos, V.; Campos, A.; Cunha, I. The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, Pollicipes pollicipes. Int. J. Mol. Sci. 2020, 21, 2524. https://doi.org/10.3390/ijms21072524 PPCIM: MS/MS raw data files comprising three biological replicates of P. pollicipes cement multicomplex obtained using label-free LC-MS high throughput proteomic analysis. LC-MS/MS system was composed by a Dionex UltiMate 3000 n-RSLC connected to an Orbitrap FusionTM TribridTM mass spectrometer (Thermo Scientific, Waltham, MA, USA).
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Three immature Pollicipes pollicipes were collected on the rocky shore of Praia da Memória beach, Portugal, 2017. The tissues collected were kept frozen at -80ºC until protein homogenization and extraction was performed in SDT buffer (2% SDS, 100mM Tris/HCl pH 7.6, 0.1M DTT). Tissues were homogenised using ultrasounds at an intensity of 60 Hz, followed by mechanical disruption using microbeads followed by incubation in SDT for 14h at 450 rpm in a thermomixer at room temperature. Samples were then centrifuged at 16000g for 20 min, the supernatant collected, the protein concentration determined by spectrophotometry at 280 nm and stored at -80ºC until further analysis. Provided lysates were incubated for 30 min with 5 mM tris(2-carboxyethyl)phosphine (TCEP) at 56 °C. The solution was brought to 10 mM TCEP and 10 mM methyl methanethiosulfonate (MMTS) for 15 min to reduce and protect cysteine residues, respectively. Protein purification, protein digestion, and peptide purification were performed according to a slightly adapted single-pot solid-phase-enhanced sample preparation (SP3) protocol. Sequencing grade trypsin was added at a ratio of 1:50 weight per weight in 50 mM HEPES, pH 8. After overnight incubation at 37°C, beads containing the digested peptides were slightly acidified using 10% FA shaken and incubated overnight at RT after raising the ACN concentration to at least 95%. Adsorbed peptides were washed once with pure acetonitrile and then air dried. Peptides were eluted in the first step with 20 µl 2% DMSO for 30 min and in the second step with 20 µl 0.065% FA, 500 mM KCl in 30% ACN for 30 min. Peptides were vacuum dried and dissolved in in 0.2% TFA / 3% ACN for subsequent ultracentrifugation (50000×g, 30 min, RT). LC-MS/MS analyses of purified and desalted peptides were performed on a Dionex UltiMate 3000 n-RSLC system connected to an Orbitrap FusionTM TribridTM mass spectrometer. Peptides of each sample were loaded onto a C18 precolumn (3 μm RP18 beads, Acclaim, 0.075 mm × 20 mm), washed for 3 min at a flow rate of 6 µl/min and separated on a C18 analytical column (3 mm, Acclaim PepMap RSLC, 0.075 mm × 50 cm, Dionex, Sunnyvale, CA, USA) at a flow rate of 200 nl/min via a linear 120 min gradient from 97% MS buffer A (0.1% FA) to 25% MS buffer B (0.1% FA, 80% ACN), followed by a 30 min gradient from 25% MS buffer B to 62% MS buffer B. The LC system was operated with the Chromeleon software (version 6.8) embedded in the Xcalibur software suite (version 3.0.63). The effluent was electro-sprayed by a stainless-steel emitter. Using the Xcalibur software, the mass spectrometer was controlled and operated in the “top speed” mode, allowing the automatic selection of as many doubly and triply charged peptides in a 3-s time window as possible, and the subsequent fragmentation of these peptides. Peptide fragmentation was carried out using the higher energy collisional dissociation mode and peptides were measured in the ion trap (HCD/IT).