Low-resolution FAIMS for increased peptide coverage in low-load and single-cell proteomics

Abstract Field Asymmetric Ion Mobility Spectrometry (FAIMS) enhances signal to noise ratio filtering ions based on their differential mobility, making it an indispensable tool for single-cell and low-input proteomics. Here, we investigate the impact of FAIMS resolution tuning via electrode temperature modulation to improve peptide identification sensitivity. We demonstrate that lowering FAIMS resolution broadens the compensation voltage window, thereby increasing ion transmission. This “low-resolution” mode significantly improves peptide identifications from a concentration range of HeLa digests by up to 18%. A weaker but still beneficial effect in peptide identifications and enhanced sensitivity could also be reproduced for single-cell samples. This leads to proteomic fingerprint shifts, resulting in distinct populations in principal component analysis from the very same cell-type in dependence on FAIMS resolution. Moreover, the increased ion counts of runs employing “low-resolution” FAIMS improve the quantitative precision of low-load measurements. These findings offer a practical optimization strategy for FAIMS-based low-input proteomics workflows, that allow for improved results by changing a single setting within the MS method without the need of any change in hardware adoption or data analysis pipelines.