Table II. Nano-LC-MS/MS instruments.
| Mass spectrometer | Q Exactive (Q-E) | Orbitrap Elite | Orbitrap Velos Pro |
|---|---|---|---|
| HPLC | Thermo EASY-nLC 1000 | Eksigent nanoLC Ultra-2D plus | Thermo EASY-nLC 1000 |
| Pre-column | Acclaim® PepMap 100 pre-column | 75 μm × 2 cm, 3 μm ReproSil-Pur 120 C18-AQ (Dr. Maisch GmbH, Germany) | |
| Analytical column | Acclaim® PepMap RSLC column | 75 μm × 15 cm, 1.9 μm ReproSil-Pur 120 C18-AQ (Dr. Maisch GmbH, Germany) | 75 μm × 15 cm, 3 μm ReproSil-Pur 120 C18-AQ (Dr. Maisch GmbH, Germany) |
| MS resolution | 70,000 | 60,000 | 60,000 |
| MS automatic gain control target | 1e6 | 1e6 | 1e6 |
| MS/MS HCD resolution | 17,500 | 15,000 | 15,000 |
| MS/MS automatic gain control target | 1e5 | HCD, 5e4; CID, 1e4 | HCD, 5e4; CID, 3e4 |
| MS/MS normalized collision energy | 35.0 | HCD, 37.5; CID, 40.0 | HCD, 36.0; CID, 40.0 |
Listed are three instrument setups and settings for MS runs. For Elite and Velos, both high-energy C-trap dissociation (HCD) and collision-induced dissociation (CID) were used for MS/MS scans.