Nontargeted Serum Lipid Profiling of Nonalcoholic Steatohepatitis by Multisegment Injection–Nonaqueous Capillary Electrophoresis–Mass Spectrometry: A Multiplexed Separation Platform for Resolving Ionic Lipids

Abstract

New methods are needed for global lipid profiling due to the complex chemical structures and diverse physicochemical properties of lipids. Herein we introduce a robust data workflow to unambiguously select lipid features from serum ether extracts by multisegment injection−nonaqueous capillary electrophoresis−mass spectrometry (MSI−NACE−MS). An iterative three-stage screening strategy is developed for nontargeted lipid analyses when using multiplexed electrophoretic separations coupled to an Orbitrap mass analyzer under negative ion mode. This approach enables the credentialing of 270 serum lipid features annotated based on their accurate mass and relative migration time, including 128 ionic lipids reliably measured (median CV ≈ 13%) in most serum samples (>75%) from nonalcoholic steatohepatitis (NASH) patients (n = 85). A mobility map is introduced to classify charged lipid classes over a wide polarity range with selectivity complementary to chromatographic separations, including lysophosphatidic acids, phosphatidylcholines, phosphatidylinositols, phosphatidylethanolamines, and nonesterified fatty acids (NEFAs). Serum lipidome profiles were also used to differentiate high-from low-risk NASH patients using a k-means clustering algorithm, where elevated circulating NEFAs (e.g., palmitic acid) were associated with increased glucose intolerance, more severe liver fibrosis, and greater disease burden. MSI−NACE−MS greatly expands the metabolome coverage of conventional aqueous-based CE−MS protocols and is a promising platform for large-scale lipidomic studies.

Metabolic Snapshot of Plasma Samples Reveals New Pathways Implicated in SARS-CoV-2 Pathogenesis

“…Even though CE-MS has some limitations, such as less migration time reproducibility, various research groups have demonstrated its value for biomarker discovery . In this sense and although CE-MS have been also applied to analyze lipids and fatty acids by using nonaqueous-CE-MS (NACE-MS), , it is the best approach to study polar and ionizable compounds such as free modified amino acids (MAAs) and “epimetabolites”, which are side products of enzyme reactions. These MAAs or the appearance of epimetabolites has been associated with important alterations in cellular, physiological, and pathological processes. − As CE-MS has been considered a powerful method to characterize unknown mechanisms of disease progression and the unique study design here included, the results here presented might fill gaps in understanding SARS-CoV-2 infection processes as well as the susceptibility to infection.…”

“…Even though CE-MS has some limitations, such as less migration time reproducibility, various research groups have demonstrated its value for biomarker discovery. 15 In this sense and although CE-MS have been also applied to analyze lipids and fatty acids by using nonaqueous-CE-MS (NACE-MS), 16,17 it is the best approach to study polar and ionizable compounds such as free modified amino acids (MAAs) and "epimetabolites", which are side products of enzyme reactions. These MAAs or the appearance of epimetabolites has been associated with important alterations in cellular, physiological, and pathological processes.…”

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…This approach has been used not only to decrease the analysis time of creatinine (by approximately 70%) [404,405] but also to inject multiple serum filtrate samples for metabolomic studies, including those of women in the last trimester of their pregnancy [406]. Other applications of this approach have been also recently described [407][408][409][410], noting that current analytical constraints of the approach include poor concentration sensitivity, potential loss in isobar/isomer resolution, sample carryover, its restriction to ionic metabolites [403], as well as the need to perform extensive method optimization to improve the compatibility with NACE protocols [411].…”

Tricky Isomers—The Evolution of Analytical Strategies to Characterize Plasmalogens and Plasmanyl Ether Lipids

“…There are both targeted and untargeted mass spectrometric approaches, and both have their specific advantages and disadvantages in terms of reliability, reproducibility, and information content. These are based on either direct infusion (shotgun) lipidomics ( Han and Gross, 2005 ; Surma et al, 2021 ) or mass spectrometric detection after different types of pre-separation, including gas or liquid chromatography ( Mawatari et al, 2007 ; Fauland et al, 2011 ; Lísa et al, 2017 ), capillary electrophoresis ( Zhang et al, 2017 ; Ly et al, 2021 ), ion mobility separation ( Vasilopoulou et al, 2020 ; Kirkwood et al, 2022 ), and supercritical fluid chromatography ( Lísa et al, 2017 ; Schoeny et al, 2020 ). In this section, we will focus on how to tackle the analytical challenge to discriminate between plasmanyl and plasmenyl lipids.…”