Robinson, H.; Molendijk, J.; Shah, A.K.; Rahman, T.; Anderson, G.J.; Hill, M.M. Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy. Biomolecules2022, 12, 1265.
Robinson, H.; Molendijk, J.; Shah, A.K.; Rahman, T.; Anderson, G.J.; Hill, M.M. Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy. Biomolecules 2022, 12, 1265.
Robinson, H.; Molendijk, J.; Shah, A.K.; Rahman, T.; Anderson, G.J.; Hill, M.M. Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy. Biomolecules2022, 12, 1265.
Robinson, H.; Molendijk, J.; Shah, A.K.; Rahman, T.; Anderson, G.J.; Hill, M.M. Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy. Biomolecules 2022, 12, 1265.
Abstract
Despite the increasing popularity of liquid chromatography-mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated method for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantification of pure lipids, however, the sample complexity and purity complex lipid extract quantification in lipidomics experiments could be impact quantification accuracy. Here, we report comprehensive assessment of the sample matrix on the accuracy of lipid quantification using Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Pure lipids are characterized by CH-and C=O-stretching vibrations on FTIR, with quantitative range of 40–3000 ng and a limit of detection of 12 ng. Sample extraction method and local baseline subtraction during FTIR spectral processing significantly impact lipid quantification by CH-stretching. To facilitate sample quality screening, we developed the Lipid Quality (LiQ) score from a spectral library of common contaminants, using a ratio of peak heights between CH-stretching vibrations maxima and the collective vibrations from amide/amine, CH-stretching minima and sugar moieties. We evaluated LiQ score as a rapid sample quality control method by comparing to total LC-MS intensity of targeted lipidomics of 107 human plasma lipid extracts. Exclusion of poor-quality samples based on LiQ score improved the correlation between FTIR and LC-MS quantification. Finally, the uncertainty of absolute quantification by FTIR was estimated using a 795 ng SPLASH LipidoMix standard to be <10%. In summary, this study identified key parameters for accurate FTIR-based quantification of complex lipid mixture, and developed a rapid workflow requiring only 1 µL of MS-ready sample and < 5 minutes for routine lipidomics sample quality and quantity assessment.
Keywords
lipids; phospholipids; sphingolipids; triglycerides; FTIR; mass spectrometry; chemical contaminants
Subject
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
