MOCCal: A Multiomic CCS Calibrator for Traveling Wave Ion Mobility Mass Spectrometry

Abstract

Ion mobility mass spectrometry (IM-MS) is a rapid, gas-phase separation technology that can resolve ions on the basis of their size-to-charge and mass-to-charge ratios. Since each class of biomolecule has a unique relationship between size and mass, IM-MS spectra of complex biological samples are organized into trendlines that each contain one type of biomolecule (i.e., lipid, peptide, metabolite). These trendlines can aid in the identification of unknown ions by providing a general classification, while more specific identifications require the conversion of IM arrival times to collision cross section (CCS) values to minimize instrument-to-instrument variability. However, the process of converting IM arrival times to CCS values varies between the different IM devices. Arrival times from traveling wave ion mobility (TWIM) devices must undergo a calibration process to obtain CCS values, which can impart biases if the calibrants are not structurally similar to the analytes. For multiomic mixtures, several different types of calibrants must be used to obtain the most accurate CCS values from TWIM platforms. Here we describe the development of a multiomic CCS calibration tool, MOCCal, to automate the assignment of unknown features to the power law calibration that provides the most accurate CCS value. MOCCal calibrates every experimental arrival time with up to three class-specific calibration curves and uses the difference (in Å2) between the calibrated TWCCSN2 value and DTCCSN2 vs m/z regression lines to determine the best calibration curve. Using real and simulated multiomic samples, we demonstrate that MOCCal provides accurately calibrated TWCCSN2 values for small molecules, lipids, and peptides.

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

“…Sample preparation. As samples, an unlabeled and a uniformly 13 C-labeled ethanolic yeast (Komagataella phaffii) extract pellet 16 were provided by Isotopic Solutions (Vienna, Austria). The yeast extract pellets were re-extracted using a lipidomics extraction based on methyl tert-butyl ether (MTBE) modified from Matyash et al 26 .…”

“…However, CCS calibrants usually have dissimilar chemical properties to analytes of interest which poses some challenges for routine 'omics workflows including lipidomics. Especially in the case of TWIM-MS lipidomics applications, it has been shown that either a calibration 12,13 or an external post-calibration correction 14 using (class-specific) lipids leads to an improved trueness of CCS values compared to DT CCS and/or published TW CCS values. Moreover, high-resolution IM analyzers including Structures for Lossless Ion Manipulation (SLIM) 15 and cyclic IMS 16 offer new possibilities for challenging lipid isomer separations for which CCS calibration provides new challenges.…”

“…To address this constraint, stable isotope labeled biomass, like the yeast strain Komagataella phaffii (also referred to as Pichia pastoris), can be employed 22 . The uniformly (U) 13 C labeled yeast extract is a well-established internal standard which covers a broad palette of U 13 C labeled lipids which, up to this point, have been primarily employed for quantification workflows [23][24][25] .…”

“…In this work, we introduce MobiLipid which utilizes a wellcharacterized fully U 13 C labeled biomass for internal standardization of IM-MS lipidomics workflows to improve CCS quality control. The provided DT CCSN2 library for U 13 C labeled lipids can be utilized for internal CCS monitoring and correction without the requirement of additional external calibration data to be measured. It can be added to routine lipidomics workflows and requires only a low number of lipids per class to be detected for successful implementation.…”

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

“…However, CCS calibrants usually have dissimilar chemical properties to analytes of interest, which poses some challenges for routine “omics” workflows, including lipidomics. Especially in the case of TWIM-MS lipidomics applications, it has been shown that either a calibration , or an external postcalibration correction using (class-specific) lipids leads to an improved trueness of CCS values compared to DT CCS and/or published TW CCS values. Moreover, high-resolution IM analyzers, including structures for lossless ion manipulation (SLIM) and cyclic IMS offer new possibilities for challenging lipid isomer separations for which a CCS calibration provides new challenges.…”