Analytical separations for lipids in complex, nonpolar lipidomes using differential mobility spectrometry

Abstract

Meibomian glands, located within the eyelids, produce a waxy, lipid-rich substance known as meibum. Meibum is thought to supply the outermost tear film lipid layer, which helps prevent the evaporation of the aqueous layer beneath it. The composition of the tear film lipid layer influences its stability (1), and changes in the ratio of lipid classes present are thought to be involved in the development of dry eye syndrome (2-4). The meibum lipidome is quite complex and is unusual in that it contains mostly neutral, nonpolar lipid classes that contain very-long (22-30 carbons) to ultra-long (>30 carbons) acyl chains. The two most abundant of these, the wax esters (WEs) and cholesteryl esters (CEs), make up around 75 mol% of total meibum lipids (5-7). Other lipids present within meibum include triacylglycerols (TGs), diacylated ,-diols (8, 9), the recently characterized (O-acyl)--hydroxy fatty acids (OAHFAs) (10, 11), and a small amount of amphiphilic lipids, including phospholipids (6,12).Two MS strategies have previously been used to identify and quantify meibomian lipids: direct infusion with tandem MS (also known as shotgun lipidomics) and LC/MS. Both approaches use soft ionization techniques, such as atmospheric pressure chemical ionization or ESI. Shotgun lipidomics has been successful in identifying and quantifying a range of lipid species within meibum (6, 12-14), but the presence of isobaric species (i.e., lipids with the same nominal mass but differing chemical composition) can Abstract Secretions from meibomian glands located within the eyelid (commonly known as meibum) are rich in nonpolar lipid classes incorporating very-long (22-30 carbons) and ultra-long (>30 carbons) acyl chains. The complex nature of the meibum lipidome and its preponderance of neutral, nonpolar lipid classes presents an analytical challenge, with typically poor chromatographic resolution, even between different lipid classes. To address this challenge, we have deployed differential mobility spectrometry (DMS)-MS to interrogate the human meibum lipidome and demonstrate near-baseline resolution of the two major nonpolar classes contained therein, namely wax esters and cholesteryl esters. Within these two lipid classes, we describe ion mobility behavior that is associated with the length of their acyl chains and location of unsaturation. This capability was exploited to profile the molecular speciation within each class and thus extend meibum lipidome coverage. Intriguingly, structuremobility relationships in these nonpolar lipids show similar trends and inflections to those previously reported for other physicochemical properties of lipids (e.g., melting point and phase-transition temperatures). Taken together, these data demonstrate that differential ion mobility provides a powerful orthoganol separation technology for the analysis of neutral lipids in complex matrices, such as meibum, and may further provide a means to predict physicochemical properties of lipids that could assist in inferring their biological function(s).

Ion mobility mass spectrometry in the omics era: Challenges and opportunities for metabolomics and lipidomics

“…FAIM‐MS has been also used in shotgun lipidomics, since it can be setup to separate each individual class by head group allowing MS detection of one lipid class at a time (Hancock et al, 2019; Lintonen et al, 2014). Separation of lipid classes by FAIM allows us to overcome the confounding factor of isobaric overlap of these molecules.…”

Lipid conformational order and the etiology of cataract and dry eye

“…Since the seminal studies of meibum composition in the 1970s ( 150 , 151 ) and ’80s ( 152 ), many techniques have been applied to separate and quantify human tear lipid and meibum moieties such as: thin-layer chromatography, high-pressure liquid chromatography, gas chromatography, and many spectrometric techniques ( 18 , 67 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 , 189 , 190 ). The focus of the current review is on relationships between meibum and TFLL composition and structure using spectroscopic techniques such as Raman, infrared, and NMR spectroscopies.…”