| Metadata |
| datasetIdentifier | PASS01156 |
| datasetType | MSMS |
| submitter | Paolo Soffientini <paolo.soffientini@ifom.eu> |
| submitter_organization | IFOM the FIRC Institute of Molecular Oncology |
| lab_head_full_name | Marco Foiani |
| lab_head_email | marco.foiani@ifom.eu |
| lab_head_organization | IFOM the FIRC Institute of Molecular Oncology |
| lab_head_country | Milan, Italy |
| datasetTag | Caf20_IP |
| datasetTitle | Caf20_IP and interactomics in Rad53-K27A, Rad53-K227A mad2delta, et no tag strain |
| publicReleaseDate | 2018-05-17 00:00:00 |
| finalizedDate | 2018-03-05 04:07:05 |
| summary | Caf20 and its interactors were co-immunoprecipitated using an anti-HA antibody covalently coupled to sepharose beads. Comparison between rad53K227A, rad53K227A mad2 and no tag cells was done. |
| contributors | Paolo Soffientini, Sophie Gay, Daniele Piccini, Marco Foiani |
| publication | Gay S, Piccini D, Bruhn C, Ricciardi S, Soffientini P, Carotenuto W, Biffo S and Foiani M.
A Mad2-mediated translational regulatory mechanism promoting S-phase cyclin synthesis controls origin firing and survival to replication stress.
Molecular Cell, submitted
|
| growth | Exponentially growing cells in YPD |
| treatment | Cells were synchronized in G1 using alpha factor and released into S-phase for 45 min in the presence of 200 mM HU. |
| extraction | Proteins were extracted using a mechanical lysis (beads) |
| separation | Mass spectrometry analysis was performed by LC–MS/MS on a quadrupole Orbitrap Q-Exactive mass spectrometer HF (Thermo Scientific) coupled with an UHPLC Easy-nLC 1000 (Thermo Scientific) with a 25 cm fused-silica emitter of 75 μm inner diameter. Both columns were packed in-house with ReproSil-Pur C18-AQ beads (Dr. Maisch Gmbh, Ammerbuch, Germany), 1.9 μm of diameter, using a high-pressure bomb loader (Proxeon, Odense, Denmark). Peptides were separeted on a linear gradient from 95% solvent A (2 % ACN, 0.1% formic acid) to 40% solvent B (80% acetonitrile, 0.1% formic acid) over 30 min and from 40% to 100% solvent B in 5 min at a constant flow rate of 0.25 μL/min with a single run time of 35 min. Samples were run in technica replicates. |
| digestion | Gel lanes were digested according to STAGE-diging protocol. The entire protocol occurs in a p1000 tip (Gilson or similar) filled at the orifice with a double C18 Empore Disk (3M, Minneapolis, MN) plug. Briefly, after Coomassie blue or Silver [16] staining, half on an entire lane was carefully cut into ~ 1 mm3 cubes and transferred into the STAGE-diging tip. These gel cubes were dehydrated with 100% acetonitrile (ACN) and rehydrated in 100 mM NH4HCO3 twice before being dehydrated by the addition of ACN. To ensure that the gel pieces do not create a sticky surface on the C18, all the solutions were added with a gel-loader tip. The removal of solutions was accomplished by centrifugation at 1800 rpm using the commercial tip box as holder. Otherwise the solutions were forced through the double C18 plug by pushing with a syringe. Reduction of protein disulfide bonds was carried out with 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3 and subsequent alkylation was performed with 55 mM iodoacetamide (IAA in complete darkness) in 100 mM NH4HCO3, at room temperature for 30 min. Both DTT and IAA were removed by centrifugation or by syringe as previously described. The gel pieces were rehydrated and dehydrated with 100 mM NH4HCO3 and ACN respectively prior to digestion. Gel pieces were rehydrated with 40 μL of Trypsin (12.5 ng/μL in 100 mM NH4HCO3, after few minutes 60 μL of NH4HCO3 were added and samples were incubated at 37 °C o/n in a commercial tip box filled by water on the bottom to ensure that buffer will not evaporate. The digestion solution was then forced through the double plug with a syringe and the flow through was collected. Samples were acidified with 100 μL of formic acid (FA) 0.1%, forced with the syringe and collected as flow-through. In this way the desalting of peptides occurs. Peptides were eluted twice by adding 100 μL of a solution composed of 80% ACN, 0.1% FA, an extra step of extraction with 100% ACN was performed and then all the eluates were dried in a Speed-Vac and resuspended in 20 μL of solvent A (2 % ACN, 0.1% formic acid). 2 and 5 μL were injected for each technical replicate on the Q-Exactive and the LTQ-FT mass spectrometers respectively. |
| acquisition | MS data were acquired using a data-dependent top 15. In the Q-Exactive HF, survey full scan MS spectra (300–1750 Th) were acquired in the Orbitrap with 70000 resolution, AGC target 1e6, IT 120 ms. For HCD spectra resolution was set to 35000, AGC target 1e5, IT 120 ms; normalized collision energy 25% and isolation width 3.0 m/z. |
| informatics | Raw data were processed with MaxQuant version 1.5.1.2. Peptides were identified from the MS–MS spectra searched against the uniprotKB_S.Cerevisiae using the Andromeda search engine, in which trypsin specificity was used with up to two missed cleavages allowed.
Cysteine carbamidomethylation was used as a fixed modification, methionine oxidation and protein amino-terminal acetylation as variable modifications. The mass deviation for MS and MS–MS peaks was set at 5 and 20 ppm respectively. The peptide and protein false discovery rates (FDRs) were set to 0.01; the minimal length required for a peptide was six amino acids; a minimum of two peptides and at least one unique peptide were required for high-confidence protein identification. The lists of identified proteins were filtered to eliminate reverse hits and known contaminants.
Label-free analysis was carried out, including a ‘match between runs’ option (time window of 5 min). A minimum ratio count of 2 was considered and the ‘LFQ intensities’, which are the intensity values normalized across the entire data set, were used.
Statistical t-test analyses were done using the Perseus program (Version 1.5.2.8) in the MaxQuant environment. For all the statistical analysis an FDR 0.05 was applied using a permutation test (500 randomizations).
Gene ontology annotation for biological process and molecular function was added. |
| instruments | Thermo Scientific Q-ecaxtive HF |
| species | Saccharomyces cerevisiae |
| massModifications | C+57.021464
variable:
M+15.9994
Prot N term 42.010 |
