Characterizing theCompensation Voltage Landscapefor Label-Free and TMTpro-Derivatized Peptides in FAIMS-Coupled Proteomics

Abstract High-field asymmetric waveform ion mobility spectrometry (FAIMS) provides a gas-phase separation dimension for LC-MS/MS proteomics, yet the selection of optimal compensation voltages (CV) remains largely empirical. To address this, we evaluated label-free and TMTpro-derivatized peptides from whole-cell lysates, systematically characterizing the transmission of over 141,000 unique tryptic peptides across a broad CV range (−10 V to −100 V). We demonstrate that FAIMS transmission is highly charge-state-dependent and modulated by mass and discrete amino acid compositions. Because single-CV methods capture less than half of the detectable proteome, we applied combinatorial modeling to evaluate multiplexed strategies. We determined that optimized 3-CV methods successfully captured ∼90% of the cumulative peptide pool identified across all tested voltages, providing evidence-based guidelines for maximizing proteome coverage in single-shot analyses.