Comparing peptide identifications by FAIMS versus quadrupole gas-phase fractionation

Abstract

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 could increase our understanding of 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 on two Thermo Eclipse Tribrid Mass Spectrometers. On both instruments, our data confirmed prior observations that FAIMS increased the number of peptides identified. We further demonstrated that the main benefit of FAIMS is due to the reduced co-isolation of persistent peptide precursor ions, which results in a decrease in chimeric spectra.

Ion Mobility–Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen

“…To overcome limited access to low abundance peptides, we utilized sample fractionation to broaden proteome coverage and allow improved detection of protease cleavage sites (neo-N-termini). While there are multiple alternative methods currently available ( 96 , 97 ), we have employed FAIMS due to its hands-off, online application for reduced sample handling, streamlined preparation, and ability to reduce co-isolation of peptide precursors ( 98 ). We validated the effectiveness of FAIMS-facilitated N-terminomics analysis by benchmarking our approach against unfractionated cell lysates, affirming robust increases in peptide identification and total proteome coverage achieved by fractionating the samples ( Figs.…”