DOI: 10.25345/c50r9mg5n
title: MassIVE MSV000096616 - Proteomics data from ROME manuscript
title: Raw data files from mass spectrometry studies for 'ROME, a novel membrane protein in vertebrates, is a key modulator of embryonal development and cancer metastasis' manuscript. Methods: For proteomics, samples were heated for 10 min at 95 degrees C. After cooling down to room temperature, 4 uL of 500 mM IAA in 5%SDS was added to a final concentration of 40 mM and incubated in the dark for 30 min. Undissolved matter was centrifuged for 8 min at 13,000 x g. The supernatant was saved and used for downstream processing using a S-Trap column (ProtiFi, LLC). Proteins were digested with sequencing-grade Lys-C/trypsin (Promega) by incubation at 37 degrees C overnight. For phosphorylation and N-glycosylation site identification, gel bands from Coomassie stained gel were treated with 20 mM DTT (Sigma) for 30 min and then 40 mM iodoacetamide (Thermo Fisher Scientific) in dark for 30 minutes. Proteins were then digested with trypsin and PNGase F at 37 degrees C overnight. Resulting peptides were eluted and dried down with a SpeedVac (Fisher Scientific). Peptides were analyzed with a nanoAcquity UPLC system coupled with Orbitrap Fusion Lumos mass spectrometer (ThermoFisher). Specifically, peptides in each sample were loaded onto a C18 Trap column (Waters AcquityUPLC M-Class Trap Symmetry C18, 100 angstrom, 5 um, 180 um x 20 mm) at 10 uL/min for 4 min. Peptides were then separated with an analytical column (Waters Acquity UPLC M-Class, peptide BEH C18 column, 300 angstrom, 1.7 um, 75 um x 150 mm) which was temperature controlled at 40 degrees C. The flow rate was set as 400 nL/min. A 90-min gradient of buffer A (2% ACN, 0.1% formic acid) and buffer B (0.1% formic acid in ACN) was used for separation: 1% buffer B at 0 min, 5% buffer B at 1 min, 22% buffer B at 60 min, 36% buffer B at 75 min, 50% buffer B at 80 min, 90% buffer B at 85 min, 90% buffer B at 90 min. Data were acquired with the Orbitrap Fusion Lumos mass spectrometer using an ion spray voltage of 2.4kV and an ion transfer temperature of 275 degrees C. Mass spectra was recorded with Xcalibur 4.0. MS parameter: OT MS: Detector Type: Orbitrap; Orbitrap Resolution: 60000; Scan Range (m/z): 375-1500; RF Lens (%): 30; AGC Target: Standard; Maximum Injection Time Mode: Auto; Microscans: 1. Charge state(s): 3-8; Exclusion duration (s): 40. Data Dependent Mode: Cycle Time; Time between Master Scans (sec): MS/MS parameter: ddMS^2 OT HCD: Isolation Mode: Quadrupole; Isolation Window (m/z): 1.6; Activation Type: HCD; HCD Collision Energy(%): 35; Detector Type: Orbitrap; Orbitrap; Resolution: 30000; Normalized AGC Target (%): 200.200. The MS raw data were processed using the Proteome Discoverer platform (version 2.4, Thermo Scientific) and Sequest HT algorithm. MS/MS data was searched against the human proteome database. Two missed tryptic cleavages were allowed, and the minimum peptide length was set to seven amino acids. Variable modifications included oxidation (M) and biotin (K). For identification of phosphorylation and glycosylation sites of ROME, variable modifications included phosphorylation (STY) and conversion of asparagine (N) to aspartic acid (D). Fixed modification included carbamidomethylation (C). MS and MS/MS ion tolerances were set at 10 ppm and 0.02 Da, respectively. The false-discovery rate (FDR) was estimated using the fixed value PSM validation.