Shotgun lipidomic analysis of human meibomian gland secretions with electrospray ionization tandem mass spectrometry

Abstract

Purpose: The purpose of this investigation was to determine the major molecular components of the lipids in normal human meibomian gland secretions (meibum).

Methods: The meibum samples were studied by direct infusion electrospray ionization (ESI), quadrupole time-of-flight mass spectrometry, and tandem mass spectrometry (MS/MS) analysis, in both positive and negative detection modes.

Results: Hundreds of peaks were detected, among which the molecular compositions and subclasses of approximately 160 major peaks were confidently identified. The compositions and subclasses of these peaks were determined from collision-induced dissociation fragmentation patterns, high-resolution and high-mass-accuracy spectra, and references of literature reports. The major peaks detected in positive mode were those of nonpolar lipids, including wax esters, cholesteryl esters, triacylglycerols, and diesters, whereas in negative mode, the major peaks detected were those of polar lipids, including free fatty acids and (O-acyl)-ω-hydroxy fatty acids.

Conclusions: The analysis of intact lipids in meibum with direct infusion ESI-MS/MS analysis has the advantages of minimal sample preparation (no chromatography or pre-separation needed), mild experimental conditions, high throughput, and high sensitivity.

Figure 1.

MS spectrum acquired in positive mode for a meibum sample. The meibum sample (∼16 μg/mL) was dissolved in chloroform/methanol mixture (2:1) with 8 μg/mL sodium iodide additive. The lipid peaks were present in sodiated form. The m/z region corresponding to each major lipid subclass is indicated with the composition of the major peaks being labeled.

Figure 2.

MS spectrum acquired in negative mode for a meibum sample. The meibum sample (∼16 μg/mL) was dissolved in chloroform/methanol mixture (2:1) with 0.1% ammonia hydroxide additive. The m/z region corresponding to each major lipid subclass is indicated with the composition of the major peaks being labeled.

Figure 3.

MS/MS spectra of ammoniated WE peaks: (a) a standard (behenyl oleate 18:1/22:0, ∼30 μg/mL) and (b) a WE mixture in meibum (total lipid concentration ∼16 μg/mL). The solutions were prepared in chloroform/methanol mixture (2:1, v/v) with 1 mM ammonium acetate additive. The WE mixtures in meibum are composed of species with the composition of fatty acid/fatty alcohol moieties being 18:1/22:0 and 16:1/24:0.

Figure 4.

MS/MS spectra of CE peaks: (a) a standard (cholesteryl nervonate) and (b) a CE in meibum. The two working solutions were prepared from diluting the stock solutions. The final concentrations of cholesteryl nervonate in (a) and the total lipids in (b) were 2 to 3 μg/mL. The final solutions contained 96:4 (v/v) methanol/chloroform with 7 to 8 μg/mL sodium iodide additives.

Figure 5.

MS/MS spectra of sodiated TG peaks in a meibum sample. The precursor ion in (a) is composed of at least two TG species with the compositions of 18:1/18:1/16:0 and 18:1/18:0/16:1 (the order may not be necessarily corresponding to the actual compositions), while that in (b) is a triolein peak. The meibum solution was the same as that in Figure 4.

Figure 6.

MS/MS spectra of ammoniated ω type I-St DE peaks. The proposed composition for the two precursor ions are shown. The meibum solution was the same as that in Figure 3.

Figure 7.

MS/MS spectra of ammoniated α,ω type II DE peaks. The proposed composition for the two precursor ions is shown. The meibum solution was the same as that in Figure 3.

Figure 8.

MS/MS spectra of three (O-acyl)-ω-hydroxy fatty acid peaks in negative mode. The experimental condition is the same as described in Figure 2.