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Chunk #52 — Methods — Proteomics — LC-MS/MS — TMT-based analysis

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Common genetic variation drives molecular heterogeneity in human iPSCs.
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yes

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5% of the material was analysed using an orbitrap fusion tribrid mass spectrometer (Thermo Scientific), equipped with a Dionex ultra high-pressure liquid chromatography system (nano RSLC). RP-LC was performed using a Dionex RSLC nano HPLC (Thermo Scientific). Peptides were injected onto a 75 μm × 2 cm PepMap-C18 pre-column and resolved on a 75 μm × 50 cm RP- C18 EASY-Spray temperature controlled integrated column-emitter (Thermo), using a four-hour multistep gradient from 5% B to 35% B with a constant flow of 200 nL min-1. The mobile phases were: 2% ACN incorporating 0.1% FA (Solvent A) and 80% ACN incorporating 0.1% FA (Solvent B). The spray was initiated by applying 2.5 kV to the EASY-Spray emitter and the data were acquired under the control of Xcalibur software in a data dependent mode using top speed and 4 s duration per cycle. The survey scan is acquired in the orbitrap covering the m/z range from 400 to 1400 Th, with a mass resolution of 120,000 and an automatic gain control (AGC) target of 2.0 e5 ions. The most intense ions were