Cross-Platform Consistency of FAIMS Compensation Voltage Optimization in Modern Orbitrap Mass Spectrometers

Abstract

High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) enhances peptide identification in LC-MS proteomics, but whether optimal compensation voltages (CVs) are transferable between mass spectrometry platforms remains unclear. To address this gap, we used the Pierce Peptide Retention Time Calibration (PRTC) mixture comprised of 15 synthetic peptides and analyzed the CV transmission profiles across a wide voltage range (−85 to −25 V). We systematically evaluated FAIMS CV profiles across three modern Orbitrap mass spectrometers (Astral, Ascend, and Exploris 480), each equipped with different ionization sources and chromatographic systems. Across the three platforms, optimal CVs were conserved within a narrow tolerance (typically ≤5 V), supporting CV portability for routine method transfer under the conditions evaluated. Additionally, we observed that interplatform CV variability was greatest for peptides with strongly negative optimal CVs, consistent with the greater conformational sensitivity of higher charge-density ions under high-field FAIMS conditions. Together, these results suggest that FAIMS CV requirements are predominantly peptide-intrinsic properties under typical bottom-up proteomics conditions and support the use of the PRTC mixture as a practical standard for routine FAIMS calibration and cross-platform quality control in proteomics laboratories.