MobiLipid: A Tool for Enhancing CCS Quality Control of Ion Mobility-Mass Spectrometry Lipidomics by Internal Standardization.

Ion mobility-mass spectrometry (IM-MS) offers benefits for lipidomics by obtaining IM-derived collision cross sections (CCS), a conditional property of an ion that can enhance lipid identification. While drift tube (DT) IM-MS retains a direct link to the primary experimental method to derive CCS values, other IM technologies rely solely on external CCS calibration, posing challenges due to dissimilar chemical properties between lipids and calibrants. To address this, we introduce MobiLipid, a novel tool facilitating the CCS quality control of IM-MS lipidomics workflows by internal standardization. MobiLipid utilizes a newly established DT CCS N2 library for uniformly (U) 13 C-labeled lipids, derived from a U 13 C-labeled yeast extract, containing 377 DT CCS N2 values. This automated open-source R Markdown tool enables internal monitoring and straightforward compensation for CCS N2 biases. It supports lipid class- and adduct-specific CCS corrections, requiring only three U 13 C-labeled lipids per lipid class-adduct combination across 10 lipid classes without requiring additional external measurements. The applicability of MobiLipid is demonstrated for trapped IM (TIM)-MS measurements of an unlabeled yeast extract spiked with U 13 C-labeled lipids. Monitoring the CCS N2 biases of TIM CCS N2 values compared to DT CCS N2 library entries utilizing MobiLipid resulted in mean absolute biases of 0.78% and 0.33% in positive and negative ionization mode, respectively. By applying the CCS correction integrated into the tool for the exemplary data set, the mean absolute CCS N2 biases of 10 lipid classes could be reduced to approximately 0%.