Table 1.
LC-MS/MS parameters performed for this study in the five core facilities.
| Parameter | TripleTOF 5600 #1 |
TripleTOF 5600 #2 |
LTQ-Orbitrap Velos |
Q Exactive | Orbitrap Elite |
|---|---|---|---|---|---|
| LC | cHiPLC Nanoflex | cHiPLC Nanoflex | UPLC | UPLC | nanoHPLC |
| Model | Eksigent | Eksigent 2D ultra plus |
Waters nanoACQUITY |
Waters nanoACQUITY |
Proxeon nEasy II |
| Peak width at half height |
17 sec | variable | <30 sec | 6 sec | variable |
| Trap | Nano cHiPLC Trap column 200 μm i.d. × 0.5 mm Eksigent 3 um (120Å) C18 |
Nano cHiPLC Trap column 200 μm i.d. × 0.5 mm Eksigent 3 um (120Å) C18 |
Integrafrit, 100 μm i.d. × 5cm of 5 μm Magic C18AQ |
Homemade 5 μm (200Å) C18 |
Homemade 5 μm (200Å) Magic C18 |
| Analytical column model |
Nano cHiPLC column Eksigent 75 μm i.d. |
Nano cHiPLC column Eksigent 75 μm i.d. |
Picofrit, 75 μm i.d. 15 μm tip |
Homemade 75 μm i.d. |
Homemade 75 μm i.d. |
| Length | 15 cm bed | 15 cm bed | 20 cm bed | 25 cm bed | 25 cm bed |
| Resin type | ChromXP C18-CL C18 on silica |
ChromXP C18-CL C18 on silica |
Magic C18AQ silica |
Magic C18AQ silica |
Magic C18AQ silica |
| Particle size | 3 μm (120Å) | 3 μm (120Å) | 3 μm (100Å) | 3 μm (100Å) | 5 μm (100Å) |
| Average pressure (psi) |
1500 | 1800 | 2500 | 3500 | 1810 |
| Sample injected | Variable volume for 200 ng |
10 μL, about < 1000 ng |
15/35 μL about < 1200 ng |
4.5 μL, about 400 ng |
12 μL, about < 1200 ng |
| Gradient length for data acquisition |
120 min | 120 min | 90 min | 90 min | 90 min |
| LC flow rate | 300 nL/min | 300 nL/min | 200 nL/min | 300 nL/min | 300 nL/min |
| Survey scan resolution |
~30,000 | ~30,000 | 30,000 | 70,000 | 120,000 |
| Survey scan m/z range |
400-1250 | 400-1250 | 395-1600 | 300-1750 | 400-1800 |
| Survey scan length | 250 msec | 250 msec | ~250 msec | Max 120 msec | 250 msec |
| Isotope exclusion ON | No | No | Yes | Yes | No |
| Fragmentation method |
CID | CID | HCD | HCD | HCD |
| Charge for MS/MS | +2 to +5 | +2 to +5 | +1 or higher | +2 to +7 | +2 or higher |
| Dynamic exclusion | 10 sec after 1 occurrence |
25 sec | Yes, 60 sec repeat count 1 |
30 sec | 100 sec |
| Max. # of MS/MS scans per period |
20 | 50 | 10 | 12 | 10 |
| MS/MS Scan Length | 100 msec | Variable for 2.8 sec period |
Max ion time 250 msec |
Max ion time 120 msec |
250 msec |
| Parent ion selection window |
0.7 Da | 0.7 Da | 1.0 Da | 1.2 Da | 1.2 Da |
| MS/MS scan resolution set |
>19,000 | ~15,000 | 7500 | 35,000 | 15,000 |
| Maximum MS/MS Fragment m/z |
2000 | 2000 | Variable | 1750 | Variable |
| Minimum MS/MS Fragment m/z |
100 | 65 | 100 | 100 | 100 |