| Metadata |
| datasetIdentifier | PASS01464 |
| datasetType | MSMS |
| submitter | Angela Bachi <angela.bachi@ifom.eu> |
| submitter_organization | IFOM |
| lab_head_full_name | |
| lab_head_email | Angela Bachi |
| lab_head_organization | IFOM |
| lab_head_country | Italy |
| datasetTag | BirIRSP53 |
| datasetTitle | Proteomic analysis of BirIRSP53 in HeLa cells |
| publicReleaseDate | 2020-05-18 00:00:00 |
| finalizedDate | 2020-05-14 23:37:03 |
| summary | Proteomic analysis of BirIRSP53 in HeLa cells, SILAC experiments Forward and Reverse |
| contributors | Sara Bisi, Andrea Disanza, Angela Cattaneo, Angela Bachi, Giorgio Scita |
| publication | Bisi et al. IRSp53 shapes the plasma membrane and controls polarized transport at nascent lumens during epithelia morphogenesis, submitted |
| growth | |
| treatment | |
| extraction | |
| separation | Protein separation by SDS-Page.
Peptide separation: 5µl of each digested sample from the forward and reverse experiments were loaded on a nLC–ESI–MS/MS quadrupole Orbitrap QExactive mass spectrometer (Thermo Fisher Scientific). Peptides were separated 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 3 min at a constant flow rate of 0.25 µl/min on UHPLC Easy-nLC 1000 (Thermo Scientific) where the LC system was connected to a 25-cm fused-silica emitter of 75 µm inner diameter (New Objective, Inc. Woburn, MA, USA), packed in-house with ReproSil-Pur C18-AQ 1.9 µm beads (Dr Maisch Gmbh, Ammerbuch, Germany). |
| digestion | Gel slices were excised from the gel sampling the entire length of the lanes, reduced with 10mM Dithiothreitol (DTT), alkylated with 55mM Iodoacetamide (IAA) and finally digested overnight with Trypsin. After acidification, peptide mixtures were concentrated and desalted on homemade StageTips µC18, ), dried in a Speed- Vac and resuspended in 12-15µL of solvent A (2 % ACN, 0.1% FA). |
| acquisition | MS data were acquired using a data-dependent top 12 method for HCD fragmentation. Survey full scan MS spectra (300–1650 Th) were acquired in the Orbitrap with 70000 resolution, AGC target 3e6, IT 60 ms.
For HCD spectra, resolution was set to 17 500 at m/z 200, AGC target 1e5, IT 120 ms; Normalized Collision energy 25% and isolation with 2.0 m/z. Technical replicates were conducted on the LC–MS-MS part of the analysis. |
| informatics | Raw data were processed with MaxQuant ver. 1.4.1.2. Peptides were identified from the MS/MS spectra searched against the uniprot_human_2014_10 database, trypsin specificity and up to two missed cleavages. Cysteine carbamidomethylation was used as fixed modification, methionine oxidation and protein N-terminal acetylation as variable modifications. Mass deviation for MS/MS peaks was set at 20 ppm. The peptides and protein FDR 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. Identified proteins were filtered to eliminate reverse hits and known contaminants. For quantitative analysis, “re-quantify” and “second peptide” options were selected. |
| instruments | Thermo Fisher Scientific LC–ESI–MS-MS quadrupole Orbitrap QExactive |
| species | Human |
| massModifications | static: C+57.021464, variable: K+8.014199, R+10.008269, M+ 15.994915, Protein N-term+ 42.010565 |
