FAIMS Pro Interface Support - Getting Started

The readback measurements for the FAIMS parameters are on the Tune status panel and in the raw data file. The following parameters are available to the user:

• CV value
• DV value
• Entrance Plate voltage
• FAIMS Gas flow
• Cooling gas flow
• Inner electrode temperature
• Outer electrode temperature by heater 1 and heater 2

No, you must complete calibration without the FAIMS attached

No, only when you change the ion source type (e.g. from H-ESI to Easy-Nano).

The CV range for tryptic peptides is between -30 V to -120 V. The resolution of FAIMS Pro in standard mode for maximal signal is a 15-20 CV window. Trypsin-digested peptides, which can be TMT-labeled, are enriched at CV values from -40 V to -80 V. To maintain optimal duty cycle, it is recommended that customers use 1 CV setting per gradient hour. Furthermore, the largest improvements in TMT quantitation accuracy occur when using 3 CV settings in a single analytical run. Therefore, it is suggested to use a three-hour gradient, switching through CV values of approximately -40 V, -60 V, and -80 V when using FAIMS Pro for TMT quantitation, or to maximize peptide ID.

High purity gas (99.5%)

References:

1. Hebert et al. Anal. Chem. 2018, 90, 9529−9537
2. Pfammatter et al. Molecular & Cellular Proteomics 17: 2051–2067, 2018. DOI: 10.1074/mcp.TIR118.000862
3. Schweppe et al. Anal. Chem. 23 Jan 2019 DOI: 10.1021/acs.analchem.8b05399

Setting FAIMS to ‘High Resolution Mode’ establishes a temperature gradient between the inner and outer electrode. This produces a peak narrowing (FWHM) electric field inside the analytical gap, which results in increased resolution. Whether or not there is a benefit to running samples in high resolution mode depends on the application. For untargeted proteomics applications, it is suggested to use standard resolution because this mode maximizes transmission and provides sufficient resolution to distinguish between singly charged and multiply charged species. High resolution mode can be beneficial for certain targeted applications. For example, if an interference optimizes at a CV value similar to that of the target analyte, increased resolution might help in separating the interference.

When FAIMS Pro is installed, but in ‘off’ mode, all ions are transmitted through the FAIMS device. This mode reduces ion transmission compared to when FAIMS voltages are on and optimal CV values are used. It is recommended only to assess spray stability.

The CV value reflects the difference between the potentials at the outer and the inner FAIMS electrode, and is set to 0 V by default. At a CV value of 0 V, a subset of ions will be transmitted through the FAIMS electrodes. Depending on the application, using a CV value of 0 might be useful to select ions of certain mobility and transmit them through the FAIMS device.

Ensure the sealing O-ring is seated in the correct position.

The large O-ring on the back-side of FAIMS (facing the mass spectrometer, see pictures below) needs to be in the correct position before installing FAIMS onto a mass spectrometer. It is recommended to do a quick check before the install. If the O-ring has slipped out of the recess, it may get damaged and/or cause a leak.

Other O-rings to check:

It is recommended to perform a leak check every time FAIMS is installed onto a mass spectrometer. Install FAIMS onto a mass spectrometer. Put a gloved finger on the entrance plate orifice. The source pressure / fore vacuum pressure should drop below the following value, which is specific to the mass spectrometer type used: On all Orbitrap Tribrid instruments, as well as all TSQ models, the source pressure should drop below 0.2 Torr. On the Orbitrap Exploris 480, the fore vacuum pressure should drop below 0.3 mbar. Note that the most accurate pressure readings are achieved when the ion transfer tube is still hot. A cool ion transfer tube may result in higher pressure readings. If the pressure does not drop below the respective value, turn FAIMS off, remove it from the mass spectrometer, and check the 3 O-rings that seal the electrodes to the mass spectrometer as described above.

The position of the emitter tip is critical for robust performance. The system is usually more robust and delivers consistent spray stability when the tip is very close to the orifice of the entrance plate. For best results, the tip of the emitter should be no more than 1-2 mm from the entrance plate. This is critical to increase robustness because the closer the spray tip, the better spray stability, and the lower the chance of droplet formation which can lead to DV instabilities. Place the emitter on the center of the x-axis and just below center on the y-axis. Keep in mind some emitters will optimize slightly off this recommendation. However, this is a good starting point.