Integration of Segmented Ion Fractionation and Differential Ion Mobility on a Q‑Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer

High-field
asymmetric waveform ion mobility spectrometry (FAIMS)
has gained popularity in the proteomics field for its capability to
improve mass spectrometry sensitivity and to decrease peptide co-fragmentation.
The recent implementation of FAIMS on Tribrid Orbitrap instruments
enhanced proteome coverage and increased the precision of quantitative
measurements. However, the FAIMS interface has not been available
on older generation Orbitrap mass spectrometers such as the Q-Exactive.
Here, we report the integration of the FAIMS Pro device with embedded
electrical and gas connections to a Q-Exactive HF mass spectrometer.
Proteomic experiments performed on HeLa tryptic digests with the modified
mass spectrometer improved signal to noise and reduced interfering
ions, resulting in an increase of 42% in peptide identification. FAIMS
was also combined with segmented ion fractionation where 100 m/z windows were obtained in turn to further
increase the depth of proteome analysis by reducing the proportion
of chimeric MS/MS spectra from 50 to 27%. We also demonstrate the
application of FAIMS to improve quantitative measurements when using
isobaric peptide labeling. FAIMS experiments performed on a two-proteome
model revealed that FAIMS Pro provided a 65% improvement in quantification
accuracy compared to conventional LC–MS/MS experiments.