Comparing peptide identifications by FAIMS versus quadrupole gas phase fractionation

Gas phase fractionation techniques have become popular in the field of mass spectrometry to improve results without using more sample. High field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to liquid chromatography-mass spectrometry (LC-MS) has been shown to increase peptide and protein detections compared to LC-MS/MS alone. However, FAIMS has not been compared to other methods of gas phase fractionation, such as quadrupole gas phase fractionation, which leaves uncertainty about the mechanisms of improvement. The goal of this work was to assess whether FAIMS improves peptide identifications because 1) gas phase fractionation enables the analysis of less abundant signals by excluding more abundant precursors from filling the ion trap, 2) the use of FAIMS reduces co-isolation of peptides during the MS/MS process resulting in a reduction of chimeric spectra, or 3) a combination of both. To investigate these hypotheses, pooled human brain tissue samples were measured in triplicate using FAIMS gas phase fractionation, quadrupole gas phase fractionation, or no gas phase fractionation. To confirm the results, the experiment was reproduced on another instrument. On both instruments, FAIMS gas phase fractionation reduced co-isolation of peptides and generated fewer chimeric spectra even though it was less efficient at transmitting ions than quadrupole gas phase fractionation. This suggests that improvements can be made to the DDA acquisition process by excluding peptides for MS/MS that have more than one peptide isotope distribution in the isolation window.