Analysis of FAIMS for the Study of Affinity-Purified Protein Complexes Using the Orbitrap Ascend Tribrid Mass Spectrometer

Abstract In this study, we analyzed the combination of affinity purification mass spectrometry (AP-MS) with high-field asymmetric waveform ion mobility spectrometry (FAIMS), integrated between nanoLC-MS and an Orbitrap Ascend Tribrid Mass Spectrometer. Our primary objective was to evaluate the application of the FAIMS interface for detecting affinity purified SAP25 protein complexes with enhanced sensitivity and robustness. As a result, we observed that nanoLC-FAIMS-MS (with FAIMS) significantly improved the sensitivity and detection limits at the protein level, peptide level and significantly reduced chemical contaminants compared to nanoLC-MS alone without FAIMS (No FAIMS). This FAIMS configuration resulted in 42% and 92% increases for the total proteins and unique proteins, respectively, and 44% and 88% increases for total peptides and unique peptides compared to the No FAIMS configuration. Our in-depth comparison of FAIMS and No FAIMS shows that FAIMS outperforms by significantly reducing the missing value by <15% in datasets and plays a significant role in filtering chemical contaminants. Our findings highlight the potential of FAIMS with Orbitrap Ascend Tribrid Mass Spectrometer to enhance the depth of AP-MS analysis. The data were deposited with the MASSIVE repository with the identifier MSV000096548. Competing Interest Statement The authors have declared no competing interest.