Table 1.
Parameters for LC-MS/MS analysis
| DDA | MS2-MS3 | EthcD | |
|---|---|---|---|
| LC parameters | |||
| Mobile phase A | 0.1% Formic acid | 0.1% Formic acid | 0.1% Formic acid |
| Mobile phase B | 80% ACN, 0.1% formic acid | 80% ACN, 0.1% formic acid | 80% ACN, 0.1% formic acid |
| Trapping column | Dionex (2 cm × 100 μm id) Acclaim Pepmap C18, 5 μm, 100 Å | Dionex (2 cm × 100 μm id) Acclaim Pepmap C18, 5 μm, 100 Å | Dionex (2 cm × 100 μm id) Acclaim Pepmap C18, 5 μm, 100 Å |
| Analytical column | Dionex (25 cm × 75 μm id) Acclaim Pepmap C18, 2 μm, 100 Å | Dionex (25 cm × 75 μm id) Acclaim Pepmap C18, 2 μm, 100 Å | Dionex (25 cm × 75 μm id) Acclaim Pepmap C18, 2 μm, 100 Å |
| Flow rate: trapping, loading time | 5 μL/min, 5 min | 5 μL/min, 5 min | 5 μL/min, 5 min |
| Flow rate: analytical | 0.3 μL/min | 0.3 μL/min | 0.3 μL/min |
| Column oven temperature | 36°C | 36°C | 36°C |
| Gradient | 0 min, 2% B 5 min, 2% B 110 min, 35% B 120 min, 90% B 127 min, 90% B 129 min, 2% B 140 min, 2% B |
0 min, 2% B 5 min, 2% B 110 min, 35% B 120 min, 90% B 127 min, 90% B 129 min, 2% B 140 min, 2% B |
0 min, 2% B 5 min, 2%B 110 min, 35% B 120 min, 90% B 127 min, 90% B 129 min, 2% B 140 min, 2% B |
| MS1 parameters | |||
| Spray voltage | 2.3 kV | 2.3 kV | 2.3 kV |
| MS1: detection | Orbitrap | Orbitrap | Orbitrap |
| MS1: resolution | 120,000 | 60,000 | 60,000 |
| MS1: scan range | 350–1,500 Da | 350–1,500 Da | 350–1,500 Da |
| MS1: mode | Profile | Profile | Profile |
| MS1: AGC target | 4.0 e5 | 4.0 e5 | 4.0 e5 |
| Maximum injection time | 50 msec | 50 msec | 50 msec |
| MS2: CID parameters | |||
| MS2: charge states | 2–7 | 4–8 | 4–8 |
| MS2: cycle time | 3 s | 5 s | 5 s |
| MS2: isolation window | 0.7 | 1.6 Da | 1.6 Da |
| MS2 fragmentation method | CID | CID | CID |
| MS2: collision energy | 35% | 25% | 25% |
| MS2: ETD activation | False | False | False |
| MS2: detection | Ion trap | Orbitrap | Orbitrap |
| MS2: resolution | Unit | 30,000 | 30,000 |
| MS2: AGC target | 3.0 e3 | 5.0 e4 | 5.0 e4 |
| MS2: mode | Centroid | Centroid | Profile |
| Maximum injection time | 300 msec | 100 msec | 100 msec |
| Dynamic exclusion | Enabled | Enabled | Enabled |
| Exclude after n times | 1 | 1 | 1 |
| Exclusion duration | 60 s | 60 s | 60 s |
| MS2: Ethcd parameters | |||
| MS2: charge States | – | – | 4–10 |
| MS2: cycle time | – | – | 5 s |
| MS2: isolation window | – | – | 1.6 Da |
| MS2 fragmentation method | – | – | ETD |
| MS2: ETD activation | – | – | True |
| ETD: collision energy | – | – | 15% |
| MS2: detection | – | – | Orbitrap |
| MS2: resolution | – | – | 30,000 |
| MS2: AGC Target | – | – | 1.0 e5 |
| MS2: mode | – | – | Centroid |
| Maximum injection time | – | – | 120 msec |
| Dynamic exclusion | – | – | Enabled |
| Exclude after n times | – | – | 1 |
| Exclusion duration | – | – | 60 s |
| MS3: parameters | DDA | MS2-MS3 | EthcD |
| Target mass difference | – | 31.9721 Da | – |
| Ions must be same charge | – | Yes | – |
| MS2: charge states | – | 2–6 | – |
| MS2: scans | – | 4 | – |
| MS2: isolation window | – | 2.0 Da | – |
| MS2 fragmentation method | – | HCD | – |
| MS2: collision energy | – | 30% | – |
| MS2: ETD activation | – | False | – |
| MS2: detection | – | Ion trap | – |
| MS2: resolution | – | Nominal | – |
| MS2: AGC target | – | 2.0 e4 | – |
| MS2: mode | – | Centroid | – |
| Maximum injection time | – | 150 s | – |