Evaluation of Collision Cross Section Calibrants for Structural Analysis of Lipids by Traveling Wave Ion Mobility-Mass Spectrometry

Abstract

Collision cross section (CCS) measurement of lipids using traveling wave ion mobility-mass spectrometry (TWIM-MS) is of high interest to the lipidomics field. However, currently available calibrants for CCS measurement using TWIM are predominantly peptides that display quite different physical properties and gas-phase conformations from lipids, which could lead to large CCS calibration errors for lipids. Here we report the direct CCS measurement of a series of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in nitrogen using a drift tube ion mobility (DTIM) instrument and an evaluation of the accuracy and reproducibility of PCs and PEs as CCS calibrants for phospholipids against different classes of calibrants, including polyalanine (PolyAla), tetraalkylammonium salts (TAA), and hexakis(fluoroalkoxy)phosphazines (HFAP), in both positive and negative modes in TWIM-MS analysis. We demonstrate that structurally mismatched calibrants lead to larger errors in calibrated CCS values while the structurally matched calibrants, PCs and PEs, gave highly accurate and reproducible CCS values at different traveling wave parameters. Using the lipid calibrants, the majority of the CCS values of several classes of phospholipids measured by TWIM are within 2% error of the CCS values measured by DTIM. The development of phospholipid CCS calibrants will enable high-accuracy structural studies of lipids and add an additional level of validation in the assignment of identifications in untargeted lipidomics experiments.

Assessment of altered lipid homeostasis by HILIC-ion mobility-mass spectrometry-based lipidomics

“…However, our CCS values tended to be smaller (average of -3.7% and -4.4% different in positive and negative modes, respectively) than previously published lipid CCS values calibrated with nonlipid calibrants, such as poly-DL-alanine (50,51). This observation is consistent with trends we observed for calibration with lipid standards versus calibration with poly-DL-alanine (36).…”

“…Calibrants that are of similar physical properties to the analytes are desired to achieve the highest accuracy of CCS measurements in TWIM. Recently, we established a series of phospholipid calibrants for measurement of lipid CCSs using TWIM, which gave CCS values that are within 2% of the values measured on DTIM (close to the errors of DTIM measurements) (36). The commercial TWIM platform is well integrated with different chromatographic separation techniques and MS analysis and is used in this study.…”

“…Mass calibration was performed with sodium formate for the range of m/z 50-1200. Collision cross-section calibration was performed using a set of PC and PE CCS standards as previously described (36 …”

“…We have recently demonstrated that the choice of calibrant has a significant impact on the CCS values generated for phospholipids on TWIM-MS platforms (36). The lipid CCS values reported here were generated from lipid standard CCS calibrants describe previously (36).…”

“…The lipid CCS values reported here were generated from lipid standard CCS calibrants describe previously (36). The difference between the calibrated CCSs of PEs and PCs identified in the lipid mix and the drift tube CCS of the corresponding PE and PC calibration standards was less than 1% in both positive and negative modes.…”