Field Asymmetric Ion Mobility Spectrometry (FAIMS) is widely used in proteomics to enhance selectivity and reduce chemical noise, but traditional high-resolution settings limit peptide coverage due to narrow compensation voltage windows that filter out many peptides. This limitation is particularly problematic in single-cell proteomics where sample amounts are extremely limited and maximising peptide identifications is crucial.

Hoch et al. aimed to address this coverage limitation by investigating whether reducing FAIMS resolution through lower outer electrode temperatures could increase peptide identifications in low-load and single-cell proteomics workflows. Their experimental approach involved systematically varying the outer electrode temperature from 100°C to 80°C whilst maintaining the inner electrode at 100°C, creating a “low-resolution” FAIMS mode that broadens the compensation voltage window and allows more ions to pass through.

The team from the Mechtler lab used a Vanquish Neo coupled to an Orbitrap Astral equipped with a FAIMS Pro Duo interface and EASY-Spray source. Peptide separation was performed on an Aurora Rapid® 8×75 XT C18 UHPLC column.

This quantitative study demonstrated significant improvements in peptide identifications, with increases ranging from 6-18% across different sample loads (250 pg to 16 ng HeLa digests). The low-resolution FAIMS approach increased peptide identifications from an average of 28,063 to 35,258 in 250 pg samples whilst maintaining protein identification levels and improving quantitative precision through reduced coefficient of variation values.

The researchers demonstrated that lowering FAIMS resolution provides a practical, cost-effective enhancement for existing low-input proteomics workflows without requiring additional hardware modifications. This approach particularly benefits single-cell proteomics applications where enhanced sensitivity is crucial for detecting low-abundance proteins that might otherwise remain undetected, ultimately improving the quality and comprehensiveness of proteomic analyses in sample-limited scenarios.

Publication
bioRxiv

Authors

Dominic G Hoch, Manuel Matzinger, & Karl Mechtler;

Title

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