Computational protocol: Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

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Protocol publication

[…] In-solution and in-gel digestion mixtures were analyzed by LC/MS on an UltiMate 3000 RSLC Nano system (Thermo Fisher Scientific, Bremen, Germany) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific, Bremen, Germany). Samples were loaded onto a pre-column (C8 reversed phase, Acclaim PepMap, 300 μm * 5 mm, 5 μm, 100 Å, Thermo Fisher Scientific, Bremen, Germany) and washed with water containing 0.1% (v/v) TFA for 15 min, before the peptides were separated on the separation column (C18 reversed phase, Acclaim PepMap, 75 μm * 250 mm, 2 μm, 100 Å, Thermo Fisher Scientific, Bremen, Germany) using gradients from 1% to 35% (v/v) B (90 min), 35% to 85% (v/v) B (5 min) followed by 85% B (5 min), with solvent A: 0.1% (v/v) formic acid (FA) in water (LC-MS grade, VWR, Darmstadt, Germany) and solvent B: 0.08% (v/v) FA in acetonitrile (LC-MS grade, VWR, Darmstadt, Germany). Data were acquired using data-dependent MS/MS mode where each high-resolution full-scan in the orbitrap (m/z 300 to 2000, R = 120,000) was followed by high-resolution product ion scans in the orbitrap (collison-induced dissociation (CID), 35% normalized collision energy, R = 15000; higher energy collision-induced dissociation (HCD) 29% normalized collision energy ± 15 stepped collision energy, R = 15,000) within 5 s, starting with the most intense signal in the full-scan mass spectrum (isolation window 2 u). For BS2G-D0/D4, a charge-dependent isolation window and offset windows were employed (charge 2+: offset 1 u, isolation window 4 u; charge 3+: offset 0.66 u, isolation window 3.5 u, charge 4+: offset 0.5 u, isolation window 3 u, charge 5+: offset 0.4 u, isolation window 2.5 u, charge 6+: offset 0.33 u, isolation window 2 u, charge 7+: offset 0.28 u, isolation window 1.5 u, charge 8+: offset 0.25 u, isolation window 1 u). Dynamic exclusion (exclusion duration: 60 s, exclusion window: ±2 ppm) was enabled to allow detection of less abundant ions. Data acquisition was controlled with Xcalibur 3.0.63 (Thermo Fisher Scientific, Bremen, Germany). Peptides were identified with the Proteome Discoverer 1.4 (Thermo Fisher Scientific) using Mascot server, version 2.2. Cross-linked products were identified with the in-house software StavroX,[] version 3.4.12 and MeroX version 1.4.12.[] All cross-links were manually evaluated. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server, StavroX, MeroX
Application MS-based untargeted proteomics
Diseases Diabetes Mellitus, Metabolic Diseases, Machado-Joseph Disease, Lipid Metabolism Disorders
Chemicals Amines